Mutant subgenomic promoter library and uses thereof

ABSTRACT

Disclosed herein is a subgenomic promoter library derived from alphavirus. Also provided herein are methods of using the subgenomic promoters to produce antibodies and other molecules.

RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119(e) to United States Provisional Application Ser. No. 62/831,561, filed Apr. 9, 2019, the entire contents of which are incorporated by reference herein.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant Nos. R01 CA206218 and R01 EB025854 awarded by the National Institutes of Health (NIH). The Government has certain rights in the invention.

BACKGROUND

The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene knockout and strong overexpression. However, many applications necessitate a slight change in gene expression level different from that of a counterpart wild-type gene expression level in a specific window; this requirement can be met by using promoter libraries. Therefore, it is of interest to develop promoters with differential levels of activity to control expression levels of multiple transgenes in one construct.

SUMMARY

The present disclosure is based, at least in part, on the unexpected discovery that certain mutants of a subgenomic promoter derived from alphavirus have differential activity in driving the expression of operably linked transgenes (e.g., genes encoding therapeutic molecules or antibody). These mutant subgenomic promoters may be used to fine tune expression level of multiple transgenes from a single alphavirus replicon (e.g., increase functional antibody production).

In some aspects, the present disclosure provides an engineered subgenomic promoter library, comprising a plurality of promoters, wherein each promoter comprises a nucleotide sequence of

GACTX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀ X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉ X₄₀X₄₁X₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅X₅₆X₅₇X₅₈ X₅₉X₆₀X₆₁X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇ X₇₈X₇₉X₈₀X₈₁X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄X₉₅X₉₆ X₉₇X₉₈X₉₉X₁₀₀X₁₀₁X₀₁₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉X₁₁₀X₁₁₁ X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉X₁₂₀X₁₂₁X₁₂₂X₁₂₃X₁₂₄X₁₂₅ X₁₂₆X₁₂₇X₁₂₈X₁₂₉X₁₃₀X₁₃₁X₁₃₂X₁₃₃X₁₃₄X₁₃₅X₁₃₆X₁₃₇X₁₃₈X₁₃₉ X₁₄₀X₁₄₁X₁₄₂X₁₄₃X₁₄₄X₁₄₅X₁₄₆X₁₄₇X₁₄₈X₁₄₉X₁₅₀X₁₅₁X₁₅₂X₁₅₃ X₁₅₄X₁₅₅X₁₅₆X₁₅₇X₁₅₈X₁₅₉X₁₆₀X₁₆₁X₁₆₂X₁₆₃X₁₆₄X₁₆₅X₁₆₆X₁₆₇ X₁₆₈X₁₆₉X₁₇₀X₁₇₁X₁₇₂X₁₇₃X₁₇₄X₁₇₅X₁₇₆X₁₇₇X₁₇₈X₁₇₉X₁₈₀X₁₈₁ X₁₈₂X₁₈₃X₁₈₄X₁₈₅X₁₈₆X₁₈₇X₁₈₈X₁₈₉X₁₉₉X₂₀₀X₂₀₁X₂₀₁X₂₀₃X₂₀₄ X₂₀₅X₂₀₆X₂₀₇X₂₀₈X₂₀₉X₂₁₀X₂₁₁X₂₁₂X₂₁₃X₂₁₄X₂₁₅X₂₁₆X₂₁₇X₂₁₈ X₂₁₉X₂₂₀X₂₂₁X₂₂₂X₂₂₃X₂₂₄X₂₂₅X₂₂₆X₂₂₇X₂₂₈X₂₂₉X₂₃₀X₂₃₁X₂₃₂ X₂₃₃X₂₃₄,

wherein X₁ is absent or present, and when X₁ is present, X₁=C; wherein X₂ is absent or present, and when X₂ is present, X₂=A; wherein X₃=T or C; wherein X₄=C or G; wherein X₅=C or A; wherein X₆=C, T or A; wherein X₇=T, G or C; wherein X₈=C, T or A; wherein X₉ is absent or present, and when X₉ is present, X₉=C or A; wherein X₁₀ is absent or present, and when X₁₀ is present, X₁₀=G; wherein X₁₁ is absent or present, and when X₁₁ is present, X₁₁=T, A or G; wherein X₁₂ is absent or present, and when X₁₂ is present, X₁₂=T or C; wherein X₁₃ is absent or present, and when X₁₃ is present, X₁₃=A or C; wherein X₁₄=G, T or A; wherein X₁₅=T, A or C; wherein X₁₆=T, C or A; wherein X₁₇=A, T or C; wherein X₁₈=T, C or A; wherein X₁₉=G, C or A; wherein X₂₀=G, C or A; wherein X₂₁ is absent or present, and when X₂₁ is present, X₂₁=C or T; wherein X₂₂ is absent or present, and when X₂₂ is present, X₂₂=A; wherein X₂₃ is absent or present, and when X₂₃ is present, X₂₃=A or T; wherein X₂₄=C or T; wherein X₂₅=A, T or G; wherein X₂₆=T, A, C, or G; wherein X₂₇=G, T or A; wherein X₂₈=A, G, or T; wherein X₂₉=C, T, or G; wherein X₃₀=T, C or A; wherein X₃₁=A, C or G; wherein X₃₂=C, T, or G; wherein X₃₃=T, C or A; wherein X₃₄=C, A, G or T; wherein X₃₅=T, C, G or A; wherein X₃₆=A, T or G; wherein X₃₇ is absent or present, and when X₃₇ is present, X₃₇=T or A; wherein X₃₈ is absent or present, and when X₃₈ is present, X₃₈=C or T; wherein X₃₉ is absent or present, and when X₃₉ is present, X₃₉=A, G or T; wherein X₄₀ is absent or present, and when X₄₀ is present, X₄₀=G, A or T; wherein X₄₁ is absent or present, and when X₄₁ is present, X₄₁=C or T; wherein X₄₂=G, C or A; wherein X₄₃=C, A or T; wherein X₄₄=T, A or C; wherein X₄₅=A, T or C; wherein X₄₆=G, T, A or C; wherein X₄₇=C or T; wherein X₄₈ is absent or present, and when X₄₈ is present, X₄₈=C or A; wherein X₄₉ is absent or present, and when X₄₉ is present, X₄₉=A or T; wherein X₅₀ is absent or present, and when X₅₀ is present, X₅₀=C or T; wherein X₅₁ is absent or present, and when X₅₁ is present, X₅₁=A or T; wherein X₅₂ is absent or present, and when X₅₂ is present, X₅₂=T or A; wherein X₅₃ is absent or present, and when X₅₃ is present, X₅₃=C; wherein X₅₄ is absent or present, and when X₅₄ is present, X₅₄=T or C; wherein X₅₅ is absent or present, and when X₅₅ is present, X₅₅=T or C; wherein X₅₆ is absent or present, and when X₅₆ is present, X₅₆=C; wherein X₅₇ is absent or present, and when X₅₇ is present, X₅₇=C, T, G or A; wherein X₅₈ is absent or present, and when X₅₈ is present, X₅₈=T; wherein X₅₉ is absent or present, and when X₅₉ is present, X₅₉=A, G or C; wherein X₆₀ is absent or present, and when X₆₀ is present, X₆₀=G, A, T, C; wherein X₆₁ is absent or present, and when X₆₁ is present, X₆₁=T, A, or G; wherein X₆₂ is absent or present, and when X₆₂ is present, X₆₂=G or A; wherein X₆₃ is absent or present, and when X₆₃ is present, X₆₃=T, A, or G; wherein X₆₄ is absent or present, and when X₆₄ is present, X₆₄=T, G, C, or A; wherein X₆₅ is absent or present, and when X₆₅ is present, X₆₅=A or G; wherein X₆₆ is absent or present, and when X₆₆ is present, X₆₆=A or G; wherein X₆₇ is absent or present, and when X₆₇ is present, X₆₇=A, T, or C; wherein X₆₈ is absent or present, and when X₆₈ is present, X₆₈=T, A, C or G; wherein X₆₉ is absent or present, and when X₆₉ is present, X₆₉=C, T, G or A; wherein X₇₀ is absent or present, and when X₇₀ is present, X₇₀=A, T or G; wherein X₇₁ is absent or present, and when X₇₁ is present, X₇₁=T or A; wherein X₇₂ is absent or present, and when X₇₂ is present, X₇₂=T or C; wherein X₇₃ is absent or present, and when X₇₃ is present, X₇₃=C, A or T; wherein X₇₄ is absent or present, and when X₇₄ is present, X₇₄=A, T or C; wherein X₇₅ is absent or present, and when X₇₅ is present, X₇₅=G, A, C or T; wherein X₇₆ is absent or present, and when X₇₆ is present, X₇₆=C or T; wherein X₇₇ is absent or present, and when X₇₇ is present, X₇₇=T, G, C or A; wherein X₇₈ is absent or present, and when X₇₈ is present, X₇₈=A, T or G; wherein X₇₉ is absent or present, and when X₇₉ is present, X₇₉=C, A or G; wherein X₈₀=C or T; wherein X₈₁ is absent or present, and when X₈₁ is present, X₈₁=T or C; wherein X₂ is absent or present, and when X₈₂ is present, X₈₂=G, A or T; wherein X₈₃ is absent or present, and when X₈₃ is present, X₈₃=G; wherein X₈₄ is absent or present, and when X₈₄ is present, X₈₄=G; wherein X₈₅ is absent or present, and when X₈₅ is present, X₈₅=C; wherein X₈₆ is absent or present, and when X₈₆ is present, X₈₆-A, G or T; wherein X₈₇ is absent or present, and when X₈₇ is present, X₈₇=G or A; wherein X₈₈ is absent or present, and when X₈₈ is present, X₈₈=A or G; wherein X₈₉ is absent or present, and when X₈₉ is present, X₈₉=G; wherein X₉₀ is absent or present, and when X₉₀ is present, X₉₀=G, T, A, or C; wherein X₉₁=G, T or C; wherein X₉₂=G, T or A; wherein X₉₃=G, A or T; wherein X₉₄=C, A or T; wherein X₉₅=C, T or G; wherein X₉₆=C, A, or G; wherein X₉₇=C, T, A or G; wherein X₉₈=T, C, G or A; wherein X₉₉=A, T, G or C; wherein X₁₀₀ is absent or present, and when X₁₀₀ is present, X₁₀₀=C; wherein X₁₀₁ is absent or present, and when X₁₁₁ is present, X₁₁₁=C; wherein X₁₀₂ is absent or present, and when X₁₀₂ is present, X₁₀₂=G, T, A or C; wherein X₁₀₃ is absent or present, and when X₁₀₃ is present, X₁₀₃=G, T, or A; wherein X₁₀₄ is absent or present, and when X₁₀₄ is present, X₁₀₄=T, C or G; wherein X₁₀₅ is absent or present, and when X₁₀₅ is present, X₁₀₅=T or G; wherein X₁₀₆ is absent or present, and when X₁₀₆ is present, X₁₀₆=T, A or G; wherein X₁₀₇ is absent or present, and when X₁₀₇ is present, X₁₀₇=T, A or C; wherein X₁₀₈ is absent or present, and when X₁₀₈ is present, X₁₀₈=A, G, T or C; wherein X₁₀₉ is absent or present, and when X₁₀₉ is present, X₁₀₉=A, T or G; wherein X₁₁₀ is absent or present, and when X₁₁₀ is present, X₁₁₀=C, T or G; wherein X₁₁₁ is absent or present, and when X₁₁₁ is present, X₁₁₁=T, or C; wherein X₁₁₂ is absent or present, and when X₁₁₂ is present, X₁₁₂=C, A, T or G; wherein X₁₃ is absent or present, and when X₁₁₃ is present, X₁₃=T, A or C; wherein X₁₁₄ is absent or present, and when X₁₁₄ is present, X₁₁₄=C, T or A; wherein X₁₁₅ is absent or present, and when X₁₁₅ is present, X₁₁₅=T, A, G or C; wherein X₁₁₆ is absent or present, and when X₁₁₆ is present, X₁₁₆=A or C; wherein X₁₁₇ is absent or present, and when X₁₁₇ is present, X₁₁₇=C, T, G or A; wherein X₁₁₈ is absent or present, and when X₈ is present, X₁₁₈=G, A, C or T; wherein X₁₁₉ is absent or present, and when X₁₁₉ is present, X₁₁₉=G, C or T; wherein X₁₂₀ is absent or present, and when X₁₂₀ is present, X₁₂₀=C, A or T; wherein X₁₂₁ is absent or present, and when X₁₂₁ is present, X₁₂₁=T, A, C or G; wherein X₁₂₂ is absent or present, and when X₁₂₂ is present, X₁₂₂=A, G, C or T; wherein X₁₂₃ is absent or present, and when X₁₂₃ is present, X₁₂₃=A, G, C or T; wherein X₁₂₄ is absent or present, and when X₁₂₄ is present, X₁₂₄=C or G; wherein X₁₂₅ is absent or present, and when X₁₂₅ is present, X₁₂₅=C or T; wherein X₁₂₆ is absent or present, and when X₁₂₆ is present, X₁₂₆=T, G or C; wherein X₁₂₇ is absent or present, and when X₁₂₇ is present, X₁₂₇=G, C or T; wherein X₁₂₈ is absent or present, and when X₁₂₈ is present, X₁₂₈=G; wherein X₁₂₉ is absent or present, and when X₁₂₉ is present, X₁₂₉=T; wherein X₁₃₀ is absent or present, and when X₁₃₀ is present, X₁₃₀=C; wherein X₁₃₁ is absent or present, and when X₁₃₁ is present, X₁₃₁=A; wherein X₁₃₂ is absent or present, and when X₁₃₂ is present, X₁₃₂=T; wherein X₁₃₃ is absent or present, and when X₁₃₃ is present, X₁₃₃=C; wherein X₁₃₄ is absent or present, and when X₁₃₄ is present, X₁₃₄=A; wherein X₁₃₅ is absent or present, and when X₁₃₅ is present, X₁₃₅=A; wherein X₁₃₆ is absent or present, and when X₁₃₆ is present, X₁₃₆=T; wherein X₁₃₇ is absent or present, and when X₁₃₇ is present, X₁₃₇=C; wherein X₁₃₈ is absent or present, and when X₁₃₈ is present, X₁₃₈=T; wherein X₁₃₉ is absent or present, and when X₁₃₉ is present, X₁₃₉=C; wherein X₁₄₀ is absent or present, and when X₁₄₀ is present, X₁₄₀=A; wherein X₁₄₁ is absent or present, and when X₁₄₁ is present, X₁₄₁=C; wherein X₁₄₂ is absent or present, and when X₁₄₂ is present, X₁₄₂=G; wherein X₁₄₃ is absent or present, and when X₁₄₃ is present, X₁₄₃=T; wherein X₁₄₄ is absent or present, and when X₁₄₄ is present, X₁₄₄=C; wherein X₁₄₅ is absent or present, and when X₁₄₅ is present, X₁₄₅=C; wherein X₁₄₆ is absent or present, and when X₁₄₆ is present, X₁₄₆=A, G, C or T; wherein X₁₄₇ is absent or present, and when X₁₄₇ is present, X₁₄₇=A, T, C or G; wherein X₁₄₈ is absent or present, and when X₁₄₈ is present, X₁₄₈=T, A, C or G; wherein X₁₄₉ is absent or present, and when X₁₄₉ is present, X₁₄₉=G, C, or A; wherein X₁₅₀ is absent or present, and when X₁₅₀ is present, X₁₅₀=G, C, A or T; wherein X₁₅₁ is absent or present, and when X₁₅₁ is present, X₁₅₁=A, C or T; wherein X₁₅₂ is absent or present, and when X₁₅₂ is present, X₁₅₂=C, G or T; wherein X₁₅₃ is absent or present, and when X₁₅₃ is present, X₁₅₃=T or C; wherein X₁₅₄ is absent or present, and when X₁₅₄ is present, X₁₅₄=A, T, G or C; wherein X₁₅₅ is absent or present, and when X₁₅₅ is present, X₁₅₅=C or T; wherein X₁₅₆ is absent or present, and when X₁₅₆ is present, X₁₅₆=G, T, A or C; wherein X₁₅₇ is absent or present, and when X₁₅₇ is present, X₁₅₇=A or G; wherein X₁₅₈ is absent or present, and when X₁₅₈ is present, X₁₅₈=C or T; wherein X₁₅₉ is absent or present, and when X₁₅₉ is present, X₁₅₉=A, T or G; wherein X₁₆₀ is absent or present, and when X₁₆₀ is present, X₁₆₀=T, A, C or G; wherein X₁₆₁ is absent or present, and when X₁₆₁ is present, X₁₆₁=A or T; wherein X₁₆₂ is absent or present, and when X₁₆₂ is present, X₁₆₂=G, T or A; wherein X₁₆₃ is absent or present, and when X₁₆₃ is present, X₁₆₃=A; wherein X₁₆₄ is absent or present, and when X₁₆₄ is present, X₁₆₄=C; wherein X₁₆₅ is absent or present, and when X₁₆₅ is present, X₁₆₅=G or A; wherein X₁₆₆ is absent or present, and when X₁₆₆ is present, X₁₆₆=C; wherein X₁₆₇ is absent or present, and when X₁₆₇ is present, X₁₆₇=T; wherein X₁₆₈ is absent or present, and when X₁₆₈ is present, X₁₆₈=C; wherein X₁₆₉ is absent or present, and when X₁₆₉ is present, X₁₆₉=G or A; wherein X₁₇₀ is absent or present, and when X₁₇₀ is present, X₁₇₀=T, G or A; wherein X₁₇₁ is absent or present, and when X₁₇₁ is present, X₁₇₁=C or T; wherein X₁₇₂ is absent or present, and when X₁₇₂ is present, X₁₇₂=T or C; wherein X₁₇₃ is absent or present, and when X₁₇₃ is present, X₁₇₃=A, G or T; wherein X₁₇₄ is absent or present, and when X₁₇₄ is present, X₁₇₄=G, A or T; wherein X₁₇₅ is absent or present, and when X₁₇₅ is present, X₁₇₅=T, C or A; wherein X₁₇₆ is absent or present, and when X₁₇₆ is present, X₁₇₆=C or T; wherein X₁₇₇ is absent or present, and when X₁₇₇ is present, X₁₇₇=C, A or T; wherein X₁₇₈ is absent or present, and when X₁₇₈ is present, X₁₇₈=G, A or C; wherein X₁₇₉ is absent or present, and when X₁₇₉ is present, X₁₇₉=C, A or T; wherein X₁₈₀ is absent or present, and when X₁₈₀ is present, X₁₈₀=C or T; wherein X₁₈₁ is absent or present, and when X₁₈₁ is present, X₁₈₁=A, C, G or T; wherein X₁₈₂ is absent or present, and when X₁₈₂ is present, X₁₈₂=A, T or C; wherein X₁₈₃ is absent or present, and when X₁₈₃ is present, X₁₈₃=G, T, A, or C; wherein X₁₈₄ is absent or present, and when X₁₈₄ is present, X₁₈₄=G, C or A; wherein X₁₈₅ is absent or present, and when X₁₈₅ is present, X₁₈₅=C or T; wherein X₁₈₆ is absent or present, and when X₁₈₆ is present, X₁₈₆=C, T or A; wherein X₁₈₇ is absent or present, and when X₁₈₇ is present, X₁₈₇=A or G; wherein X₁₈₈ is absent or present, and when X₁₈₈ is present, X₁₈₈=C or T; wherein X₁₈₉ is absent or present, and when X₁₈₉ is present, X₁₈₉=C, A or T; wherein X₁₉₀ is absent or present, and when X₁₉₀ is present, X₁₉₀=A, T, A or G; wherein X₁₉₁ is absent or present, and when X₁₉₁ is present, X₁₉₁=T, G, or A; wherein X₁₉₂ is absent or present, and when X₁₉₂ is present, X₁₉₂=A or T; wherein X₁₉₃ is absent or present, and when X₁₉₃ is present, X₁₉₃=T, or A; wherein X₁₉₄ is absent or present, and when X₁₉₄ is present, X₁₉₄=A or G; wherein X₁₉₅ is absent or present, and when X₁₉₅ is present, X₁₉₅=G, A, or T; wherein X₁₉₆ is absent or present, and when X₁₉₆ is present, X₁₉₆=G or T; wherein X₁₉₇ is absent or present, and when X₁₉₇ is present, X₁₉₇=T, C, or A; wherein X₁₉₈ is absent or present, and when X₁₉₈ is present, X₁₉₈=A or G; wherein X₁₉₉ is absent or present, and when X₁₉₉ is present, X₁₉₉=T or C; wherein X₂₀₀ is absent or present, and when X₂₀₀ is present, X₂₀₀=G, T, A or C; wherein X₂₀₁ is absent or present, and when X₂₀₁ is present, X₂₀₁=G, T or A; wherein X₂₀₂ is absent or present, and when X₂₀₂ is present, X₂₀₂=G, C or A; wherein X₂₀₃ is absent or present, and when X₂₀₃ is present, X₂₀₃=C or T; wherein X₂₀₄ is absent or present, and when X₂₀₄ is present, X₂₀₄=A or T; wherein X₂₀₅ is absent or present, and when X₂₀₅ is present, X₂₀₅=A, C or G; wherein X₂₀₆ is absent or present, and when X₂₀₆ is present, X₂₀₆=C or A; wherein X₂₀₇ is absent or present, and when X₂₀₇ is present, X₂₀₇=A or G; wherein X₂₀₈ is absent or present, and when X₂₀₈ is present, X₂₀₈=C; wherein X₂₀₉ is absent or present, and when X₂₀₉ is present, X₂₀₉=C; wherein X₂₁₀ is absent or present, and when X₂₁₀ is present, X₂₁₀=C or T; wherein X₂₁₁ is absent or present, and when X₂₁₁ is present, X₂₁₁=A or G; wherein X₂₁₂ is absent or present, and when X₂₁₂ is present, X₂₁₂=C or A; wherein X₂₁₃ is absent or present, and when X₂₁₃ is present, X₂₁₃=C or A; wherein X₂₁₄ is absent or present, and when X₂₁₄ is present, X₂₁₄=G; wherein X₂₁₅ is absent or present, and when X₂₁₅ is present, X₂₁₅=A; wherein X₂₁₆ is absent or present, and when X₂₁₆ is present, X₂₁₆=G; wherein X₂₁₇ is absent or present, and when X₂₁₇ is present, X₂₁₇=C; wherein X₂₁₈ is absent or present, and when X₂₁₈ is present, X₂₁₈=G; wherein X₂₁₉ is absent or present, and when X₂₁₉ is present, X₂₁₉=C; wherein X₂₂₀ is absent or present, and when X₂₂₀ is present, X₂₂₀=T; wherein X₂₂₁ is absent or present, and when X₂₂₁ is present, X₂₂₁=T; wherein X₂₂₂ is absent or present, and when X₂₂₂ is present, X₂₂₂=C; wherein X₂₂₃ is absent or present, and when X₂₂₃ is present, X₂₂₃=G; wherein X₂₂₄ is absent or present, and when X₂₂₄ is present, X₂₂₄=T; wherein X₂₂₅ is absent or present, and when X₂₂₅ is present, X₂₂₅=C; wherein X₂₂₆ is absent or present, and when X₂₂₆ is present, X₂₂₆=G; wherein X₂₂₇ is absent or present, and when X₂₂₇ is present, X₂₂₇=A; wherein X₂₂₈ is absent or present, and when X₂₂₈ is present, X₂₂₈=G; wherein X₂₂₉ is absent or present, and when X₂₂₉ is present, X₂₂₉=G; wherein X₂₃₀ is absent or present, and when X₂₃₀ is present, X₂₃₀=C; wherein X₂₃₁ is absent or present, and when X₂₃₁ is present, X₂₃₁=C; wherein X₂₃₂ is absent or present, and when X₂₃₂ is present, X₂₃₂=A; wherein X₂₃₃ is absent or present, and when X₂₃₃ is present, X₂₃₃=C; wherein X₂₃₄ is absent or present, and when X₂₃₄ is present, X₂₃₄=C (SEQ ID NO: 81).

In some aspects, the present disclosure provides an engineered subgenomic promoter library comprising a plurality of promoters. Each promoter can comprise a nucleic acid sequence at least 70% identical to nucleic acid sequences of SEQ IDs NO: 1-74 and 81. In some embodiments, the engineered subgenomic promoter library is a subgenomic promoter derived from an alphavirus. In some embodiments, the alphavirus is Venezuela Equine Encephalitis virus, Semliki Forest virus, or Sindbis virus.

The subgenomic promoters described herein may further comprise restriction endonuclease sites at the 5′ and 3′ ends. In some embodiments, the restriction endonuclease sites at the 5′ and 3′ ends are SapI sites.

In some embodiments, the engineered subgenomic promoter library comprises engineered subgenomic promoters having differential activities.

Other aspects of the present disclosure provide engineered nucleic acids. In some embodiments, the engineered nucleic acids include a promoter selected from the engineered subgenomic promoter library and the promoter is operably linked to a transgene. In some embodiments, the transgene encodes a therapeutic molecule or a detectable molecule. In other embodiments, the transgene encodes a heavy chain or a light chain of an antibody.

Further provided herein are expression cassettes that include one or more engineered nucleic acid described herein. In some embodiments, the expression cassette is an antibody expression cassette. In some embodiments, the antibody expression cassettes include a first and a second engineered nucleic acids as described herein. In some embodiments, the first engineered nucleic acid includes a transgene encoding a heavy chain of an antibody. In some embodiments, the second engineered nucleic acid includes a transgene encoding a light chain of an antibody. In some embodiments, the antibody expression cassette comprises a first engineered nucleic acid comprising a first engineered subgenomic promoter selected from the engineered subgenomic promoter library operably linked to a first transgene, and the first transgene encodes a heavy chain of an antibody; and a second engineered nucleic acid comprising a second engineered subgenomic promoter selected from the engineered subgenomic promoter library operably linked to a second transgene, and the second transgene encodes a light chain of an antibody.

In another aspect, the disclosure provides vectors comprising the engineered nucleic acid described herein. In some embodiments, the vectors include one or more engineered nucleic acid, or one or more expression cassette described herein. In some embodiments, the vector is a plasmid, a RNA replicon, linear double stranded DNA, viral vectors, liposome or nanoparticles. In some examples, the RNA replicon is derived from an alphavirus. In one example, the one or more engineered nucleic acid is located at the subgenomic region of the RNA replicon.

In another aspect, the present disclosure also provides cells comprising the engineered nucleic acid or the expression cassette described herein are provided.

Other aspects of the invention provide methods for selecting an antibody expression cassette for optimized production of functional antibody. The methods include constructing an antibody expression cassette library comprising a plurality of antibody expression cassettes for expression of a heavy chain and a light chain of an antibody, wherein the antibody expression cassette comprises: a first engineered nucleic acid comprising a first engineered subgenomic promoter selected from the engineered subgenomic promoter library operably linked to a first transgene, and the first transgene encodes a heavy chain of an antibody; and a second engineered nucleic acid comprising a second engineered subgenomic promoter selected from the engineered subgenomic promoter library operably linked to a second transgene, and the second transgene encodes a light chain of an antibody. The methods further include delivering the plurality of engineered nucleic acids to a population of cells; culturing the cell under conditions allowing for expression of the heavy chain and the light chain of the antibody; measuring a level of functional antibody comprising the heavy chain and the light chain produced by population of cells; selecting cell(s) expressing optimal level of functional antibody; and determining the nucleic acid sequence of the subgenomic promoter in the first engineered nucleic acid and the nucleic acid sequence of the subgenomic promoter in the second engineered nucleic acid.

In another aspect, methods for producing an antibody are provided. The methods include constructing an antibody expression cassette described herein for expression of a heavy chain and a light chain of an antibody; delivering the antibody expression cassette to a population of cells or a host animal; culturing the cell or growing the host animal under conditions allowing for expression of the heavy chain and the light chain of the antibody; and harvesting the cultured host cell or culture medium or tissue from the host animal for collection of the antibody. In some embodiments, the method further includes purifying the antibody.

Also provided herein are methods for producing one or more molecules. The methods include constructing an expression cassette described herein for expression of the one or more molecules; delivering the expression cassette to a population of cells or a host animal; culturing the cell or growing the host animal under conditions allowing for expression of the one or more molecules; and harvesting the cultured host cell or culture medium or tissue from the host animal for collection of the molecule. In some embodiments, the method further includes purifying the one or more molecules. In some embodiments, the one or more molecules encoded by the expression cassette (e.g., by a transgene operably linked to a subgenomic promoter) is a detectable molecule or a therapeutic molecule.

The details of embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B are graphs showing that the subgenomic mutant promoters in the library have differential levels of activity. FIG. 1A is a chart illustrating the gating strategy for sorting cells expressing various levels of mVenus and mKate. FIG. 1B is a chart showing the varying expression strength of different subgenomic promoters selected from the subgenomic promoter library.

FIG. 2 is an illustration of the exemplary mutant subgenomic promoters in the subgenomic promoter library with annotated mutations as compared to wild type subgenomic promoter. Top to bottom, the sequences correspond to SEQ ID NOs: 80, 1, 1, 1, 1, 1, 1, 1, 19, 1, 1, 73, 31, 56, 60, 55, 50, 47, 44, 39, 27, 22, 17, 3, 64, 61, 10, 70, 29, 48, 30, 53, 32, 20, 5, 59, 37, 49, 54, 40, 16, 13, 9, 36, 28, 69, 34, 62, 18, 66, 72, 38, 35, 33, 65, 52, 15, 24, 68, 43, 4, 20, 8, 58, 46, 74, 6, 71, 7, 67, 26, 11, 25, 12, and 64.

DETAILED DESCRIPTION

The present disclosure is based, at least in part, on the unexpected discovery that certain mutants of a subgenomic promoter derived from alphavirus have differential activity in driving the expression of downstream transgenes (e.g., therapeutic molecules or antibody).

These mutant subgenomic promoters may be used to fine tune expression level of multiple transgenes from a single alphavirus replicon (e.g., increase functional antibody production).

I. Engineered Subgenomic Promoter Library

A “promoter” refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter regulates (e.g., activates or represses) expression or transcription of the nucleic acid sequence that it is operably linked to. A promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and transcription factors. Promoters may be constitutive, inducible, activatable, repressible, tissue-specific, cell type-specific, cell state-specific, or any combination thereof. In some embodiments, the promoter described herein is a subgenomic promoter.

Promoters of the present disclosure are engineered promoters. An engineered promoter is a promoter that is not “naturally occurring.” The engineered promoters of the present disclosure may be produced using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction (PCR) (see U.S. Pat. Nos. 4,683,202 and 5,928,906). The term “subgenomic promoter” is used interchangeably with “engineered subgenomic promoter” herein.

Promoters of the present disclosure are subgenomic promoters derived from an alphavirus. Many positive-sense RNA viruses, e.g., alphaviruses, produce these subgenomic mRNAs (sgRNA) as one of the common infection techniques used by these viruses and generally transcribe late viral genes. As used herein, the term “subgenome” or “subgenomic” refers to a smaller section of the whole replicon genome. Accordingly, subgenomic transcription, as used herein, refers to the transcription of one or more genes in the replicon genome but not all the genes constituting the replicon genome, which genes can be heterologous to the virus genome, such as transgenes in engineered virus genomes including the replicons described herein. Thus in some embodiments, subgenomic transcription refers to transcription of the genes of experimental or therapeutic interest, which are described elsewhere herein (e.g., antibodies). In such cases, the subgenomic promoter is a promoter used to regulate transcription of a specific heterologous gene to which the promoter is operably linked, resulting in the formation of mRNA for that gene alone.

In some embodiments, the length of an engineered subgenomic promoter is 200 nucleotides or shorter. In some embodiments, a subgenomic promoter may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 nucleotides long.

A virus is a small pathogen that is only capable of replication inside a living host cell (e.g., prokaryotic and eukaryotic cells). Outside of living cells, viruses exist as independent particles (e.g., viral particles or virions), which comprise genetic material in the form of DNA or RNA, which can be single-stranded or double-stranded. Viruses with DNA genomes are referred to as DNA viruses, and viruses with RNA genomes are referred to as RNA viruses. In some cases, the virus comprises nucleic acid-associated proteins and the combination of the virus and nucleic acid-associated proteins is referred to as nucleoprotein. In addition to the genetic material, viruses have a single or double protein coat, also known as a capsid, which facilitates attachment of the virus to a living host cell's receptors during infection and protects the genetic material of the virus from enzymatic degradation. The combination of nucleoprotein and the capsid is referred to as a nucleocapsid. In some cases, viruses have a lipid bilayer envelope, studded with virus-coded, glycosylated (trans) membrane-associated proteins. Once a virus has infected a living host cell, the virus is dependent on the living host cell to supply the machinery for its replication, and propagation thereafter. The viral genome codes for some structural proteins and non-structural regulatory proteins.

In some embodiments, the subgenomic promoter provided herein is derived from an alphavirus. Distinct from host mRNA, alphavirus replicon RNAs encode a set of four nonstructural proteins (nsPs 1-4) that are responsible both for genome replication. When engineered to include gene(s) encoding non-viral products, such as therapeutic molecules, in place of one or more of the nonstructural protein genes, alphavirus replicon RNAs provide for transcription of such non-viral products under the subgenomic promoter(s) to which the gene(s) are operably linked.

Alphaviruses are part of the Group IV Togaviridae family of viruses, possess a positive sense, single-stranded RNA genome, and are characterized by an icosahedral nucleocapsid. Other non-limiting examples of Group IV viruses include Astroviridae, Caliciviridae, Coronaviridae, Flaviviridae, Picornaviridae, Arteriviridae, and Togaviridae. The alphavirus genus includes 26 enveloped viruses that infect eukaryotes. Alphaviruses have a broad host range and are transmitted by mosquitoes and hematophagous arthropods. Non-limiting examples of alphaviruses include Venezuelan equine encephalitis virus (VEE), Semliki Forest virus (SF), Sindbis virus (SIN), Eastern Equine Encephalitis virus (EEE), Western equine encephalitis virus (WEE), Everglades virus (EVE), Mucambo virus (MUC), Pixuna virus (PIX), Semliki Forest virus (SF), Middelburg virus (MID), Chikungunya virus (CHIK), O'Nyong-Nyong virus (ONN), Ross River virus (RR), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAG), Bebaru virus (BEB), Mayaro virus (MAY), Una virus (UNA), Aura virus (AURA), Babanki virus (BAB), Highlands J virus (HJ), and Fort Morgan virus (FM).

In some instances, the alphavirus replicon is a VEE alphavirus replicon. The virion of VEE is spherical and possesses a lipid membrane with glycoprotein surface proteins spread around the outer surface. Typically, VEE has a genome of approximately 11.45 kb, excluding the 5′-terminal cap and 3′-terminal poly(A) tract, and comprises 4 nonstructural proteins (nsPs) and 5 structural proteins. The non-structural proteins include nsP1, nsP2, nsP3, and nsP4, while the structural region encodes proteins C, E3, E2, 6K, and E1. In some instances, the subgenomic promoter is a WT subgenomic promoter derived from VEE. In other instances, the subgenomic promoter is a mutant subgenomic promoter. Mutant sequences of the subgenomic promoters can be obtained by conventional methods in the art, e.g., error prone PCR. Non limiting examples of mutations of the mutant subgenomic promoters are point mutations, deletions, or insertions.

Exemplary sequence of the wild type VEE virus subgenomic promoter region (−99/+30) and a Kozak sequence (in bold face) is set forth in SEQ ID NO: 1:

(SEQ ID NO: 1) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC.

A Kozak sequence is a sequence which occurs in eukaryotic mRNA and plays a major role in the initiation of the translation process. In some embodiments, the Kozak sequence has a consensus sequence (gcc)gccRccAUGG (SEQ ID NO: 79). In this consensus sequence, AUG indicate the translation start codon, coding for Methionine; upper-case letters indicate highly conserved bases, i.e. the ‘AUGG’ sequence is constant or rarely, if ever, changes; ‘R’ indicates that a purine (adenine or guanine) is always observed at this position (with adenine being more frequent according to Kozak); a lower-case letter denotes the most common base at a position where the base can nevertheless vary; the sequence in parentheses (gcc) is of uncertain significance. Variations of the Kozak sequence are known in the art, and can be selected according to the application of interest (see, e.g., Hernadez, Conservation and Variability of the AUG Initiation Codon Context in Eukaryotes, Trends in Biochemical Sciences, Volume 44, Issue 12, December 2019, Pages 1009-1021). In some embodiments, An exemplary Kozak sequence is GCCACC (SEQ ID NO: 75). In some embodiments, the subgenomic promoter optionally comprises a Kozak sequence at the 3′ end.

In some embodiments, the mutant subgenomic promoters in the subgenomic promoter library comprises one or more mutation compared to the wild-type subgenomic promoters. In some embodiments, the subgenomic promoters in the library comprises a consensus nucleotide sequence as set forth in SEQ ID NO: 81:

GACTX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀ X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉ X₄₀X₄₁X₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅X₅₆X₅₇X₅₈ X₅₉X₆₀X₆₁X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇ X₇₈X₇₉X₈₀X₈₁X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄X₉₅X₉₆ X₉₇X₉₈X₉₉X₁₀₀X₁₀₁X₀₁₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉X₁₁₀X₁₁₁ X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉X₁₂₀X₁₂₁X₁₂₂X₁₂₃X₁₂₄X₁₂₅ X₁₂₆X₁₂₇X₁₂₈X₁₂₉X₁₃₀X₁₃₁X₁₃₂X₁₃₃X₁₃₄X₁₃₅X₁₃₆X₁₃₇X₁₃₈X₁₃₉ X₁₄₀X₁₄₁X₁₄₂X₁₄₃X₁₄₄X₁₄₅X₁₄₆X₁₄₇X₁₄₈X₁₄₉X₁₅₀X₁₅₁X₁₅₂X₁₅₃ X₁₅₄X₁₅₅X₁₅₆X₁₅₇X₁₅₈X₁₅₉X₁₆₀X₁₆₁X₁₆₂X₁₆₃X₁₆₄X₁₆₅X₁₆₆X₁₆₇ X₁₆₈X₁₆₉X₁₇₀X₁₇₁X₁₇₂X₁₇₃X₁₇₄X₁₇₅X₁₇₆X₁₇₇X₁₇₈X₁₇₉X₁₈₀X₁₈₁ X₁₈₂X₁₈₃X₁₈₄X₁₈₅X₁₈₆X₁₈₇X₁₈₈X₁₈₉X₁₉₉X₂₀₀X₂₀₁X₂₀₁X₂₀₃X₂₀₄ X₂₀₅X₂₀₆X₂₀₇X₂₀₈X₂₀₉X₂₁₀X₂₁₁X₂₁₂X₂₁₃X₂₁₄X₂₁₅X₂₁₆X₂₁₇X₂₁₈ X₂₁₉X₂₂₀X₂₂₁X₂₂₂X₂₂₃X₂₂₄X₂₂₅X₂₂₆X₂₂₇X₂₂₈X₂₂₉X₂₃₀X₂₃₁X₂₃₂ X₂₃₃X₂₃₄, in which X₁ is absent or present, and when X₁ is present, X₁=C; wherein X₂ is absent or present, and when X₂ is present, X₂=A; wherein X₃=T or C; wherein X₄=C or G; wherein X₅=C or A; wherein X₆=C, T or A; wherein X₇=T, G or C; wherein X₈=C, T or A; wherein X₉ is absent or present, and when X₉ is present, X₉=C or A; wherein X₁₀ is absent or present, and when X₁₀ is present, X₁₀=G; wherein X₁₁ is absent or present, and when X₁₁ is present, X₁₁=T, A or G; wherein X₁₂ is absent or present, and when X₁₂ is present, X₁₂=T or C; wherein X₁₃ is absent or present, and when X₁₃ is present, X₁₃=A or C; wherein X₁₄=G, T or A; wherein X₁₅=T, A or C; wherein X₁₆=T, C or A; wherein X₁₇=A, T or C; wherein X₁₈=T, C or A; wherein X₁₉=G, C or A; wherein X₂₀=G, C or A; wherein X₂₁ is absent or present, and when X₂₁ is present, X₂₁=C or T; wherein X₂₂ is absent or present, and when X₂₂ is present, X₂₂=A; wherein X₂₃ is absent or present, and when X₂₃ is present, X₂₃=A or T; wherein X₂₄=C or T; wherein X₂₅=A, T or G; wherein X₂₆=T, A, C, or G; wherein X₂₇=G, T or A; wherein X₂₈=A, G, or T; wherein X₂₉=C, T, or G; wherein X₃₀=T, C or A; wherein X₃₁=A, C or G; wherein X₃₂=C, T, or G; wherein X₃₃=T, C or A; wherein X₃₄=C, A, G or T; wherein X₃₅=T, C, G or A; wherein X₃₆=A, T or G; wherein X₃₇ is absent or present, and when X₃₇ is present, X₃₇=T or A; wherein X₃₈ is absent or present, and when X₃₈ is present, X₃₈=C or T; wherein X₃₉ is absent or present, and when X₃₉ is present, X₃₉=A, G or T; wherein X₄₀ is absent or present, and when X₄₀ is present, X₄=G, A or T; wherein X₄₁ is absent or present, and when X₄₁ is present, X₄₁=C or T; wherein X₄₂=G, C or A; wherein X₄₃=C, A or T; wherein X₄₄=T, A or C; wherein X₄₅=A, T or C; wherein X₄₆=G, T, A or C; wherein X₄₇=C or T; wherein X₄₈ is absent or present, and when X₄₈ is present, X₄₈=C or A; wherein X₄₉ is absent or present, and when X₄₉ is present, X₄₉=A or T; wherein X₅₀ is absent or present, and when X₅₀ is present, X₅₀=C or T; wherein X₅₁ is absent or present, and when X₅₁ is present, X₅₁=A or T; wherein X₅₂ is absent or present, and when X₅₂ is present, X₅₂=T or A; wherein X₅₃ is absent or present, and when X₅₃ is present, X₅₃=C; wherein X₅₄ is absent or present, and when X₅₄ is present, X₅₄=T or C; wherein X₅₅ is absent or present, and when X₅₅ is present, X₅₅=T or C; wherein X₅₆ is absent or present, and when X₅₆ is present, X₅₆=C; wherein X₅₇ is absent or present, and when X₅₇ is present, X₅₇=C, T, G or A; wherein X₅₈ is absent or present, and when X₅₈ is present, X₅₈=T; wherein X₅₉ is absent or present, and when X₅₉ is present, X₅₉=A, G or C; wherein X₆₀ is absent or present, and when X₆₀ is present, X₆₀=G, A, T, C; wherein X₆₁ is absent or present, and when X₆₁ is present, X₆₁=T, A, or G; wherein X₆₂ is absent or present, and when X₆₂ is present, X₆₂=G or A; wherein X₆₃ is absent or present, and when X₆₃ is present, X₆₃=T, A, or G; wherein X₆₄ is absent or present, and when X₆₄ is present, X₆₄=T, G, C, or A; wherein X₆₅ is absent or present, and when X₆₅ is present, X₆₅=A or G; wherein X₆₆ is absent or present, and when X₆₆ is present, X₆₆=A or G; wherein X₆₇ is absent or present, and when X₆₇ is present, X₆₇=A, T, or C; wherein X₆₈ is absent or present, and when X₆₈ is present, X₆₈=T, A, C or G; wherein X₆₉ is absent or present, and when X₆₉ is present, X₆₉=C, T, G or A; wherein X₇₀ is absent or present, and when X₇₀ is present, X₇₀=A, T or G; wherein X₇₁ is absent or present, and when X₇₁ is present, X₇₁=T or A; wherein X₇₂ is absent or present, and when X₇₂ is present, X₇₂=T or C; wherein X₇₃ is absent or present, and when X₇₃ is present, X₇₃=C, A or T; wherein X₇₄ is absent or present, and when X₇₄ is present, X₇₄=A, T or C; wherein X₇₅ is absent or present, and when X₇₅ is present, X₇₅=G, A, C or T; wherein X₇₆ is absent or present, and when X₇₆ is present, X₇₆=C or T; wherein X₇₇ is absent or present, and when X₇₇ is present, X₇₇=T, G, C or A; wherein X₇₈ is absent or present, and when X₇₈ is present, X₇₈=A, T or G; wherein X₇₉ is absent or present, and when X₇₉ is present, X₇₉=C, A or G; wherein X₈₀=C or T; wherein X₈₁ is absent or present, and when X₈₁ is present, X₈₁=T or C; wherein X₈₂ is absent or present, and when X₈₂ is present, X₈₂=G, A or T; wherein X₈₃ is absent or present, and when X₈₃ is present, X₈₃=G; wherein X₈₄ is absent or present, and when X₈₄ is present, X₈₄=G; wherein X₈₅ is absent or present, and when X₈₅ is present, X₈₅=C; wherein X₈₆ is absent or present, and when X₈₆ is present, X₈₆=A, G or T; wherein X₈₇ is absent or present, and when X₈₇ is present, X₈₇=G or A; wherein X₈₈ is absent or present, and when X₈₈ is present, X₈₈=A or G; wherein X₈₉ is absent or present, and when X₈₉ is present, X₈₉=G; wherein X₉₀ is absent or present, and when X₉₀ is present, X₉₀=G, T, A, or C; wherein X₉₁=G, T or C; wherein X₉₂=G, T or A; wherein X₉₃=G, A or T; wherein X₉₄=C, A or T; wherein X₉₅=C, T or G; wherein X₉₆=C, A, or G; wherein X₉₇=C, T, A or G; wherein X₉₈=T, C, G or A; wherein X₉₉=A, T, G or C; wherein X₁₀₀ is absent or present, and when X₁₀₀ is present, X₁₀₀=C; wherein X₁₀₁ is absent or present, and when X₁₀₁ is present, X₁₀₁=C; wherein X₁₀₂ is absent or present, and when X₁₀₂ is present, X₁₀₂=G, T, A or C; wherein X₁₀₃ is absent or present, and when X₁₀₃ is present, X₁₀₃=G, T, or A; wherein X₁₀₄ is absent or present, and when X₁₀₄ is present, X₁₀₄=T, C or G; wherein X₁₀₅ is absent or present, and when X₁₀₅ is present, X₁₀₅=T or G; wherein X₁₀₆ is absent or present, and when X₁₀₆ is present, X₁₀₆=T, A or G; wherein X₁₀₇ is absent or present, and when X₁₀₇ is present, X₁₀₇=T, A or C; wherein X₁₀₈ is absent or present, and when X₁₀₈ is present, X₁₀₈=A, G, T or C; wherein X₁₀₉ is absent or present, and when X₁₀₉ is present, X₁₀₉=A, T or G; wherein X₁₁₀ is absent or present, and when X₁₁₀ is present, X₁₁₀=C, T or G; wherein X₁₁₁ is absent or present, and when X₁₁₁ is present, X₁₁₁=T, or C; wherein X₁₁₂ is absent or present, and when X₁₁₂ is present, X₁₁₂=C, A, T or G; wherein X₁₁₃ is absent or present, and when X₁₁₃ is present, X₁₁₃=T, A or C; wherein X₁₁₄ is absent or present, and when X₁₁₄ is present, X₁₁₄=C, T or A; wherein X₁₁₅ is absent or present, and when X₁₁₅ is present, X₁₁₅=T, A, G or C; wherein X₁₁₆ is absent or present, and when X₁₁₆ is present, X₁₁₆=A or C; wherein X₁₁₇ is absent or present, and when X₁₁₇ is present, X₁₁₇=C, T, G or A; wherein X₁₁₈ is absent or present, and when X₁₁₈ is present, X₁₁₈=G, A, C or T; wherein X₁₁₉ is absent or present, and when X₁₁₉ is present, X₁₁₉=G, C or T; wherein X₁₂₀ is absent or present, and when X₁₂₀ is present, X₁₂₀=C, A or T; wherein X₁₂₁ is absent or present, and when X₁₂₁ is present, X₁₂₁=T, A, C or G; wherein X₁₂₂ is absent or present, and when X₁₂₂ is present, X₁₂₂=A, G, C or T; wherein X₁₂₃ is absent or present, and when X₁₂₃ is present, X₁₂₃=A, G, C or T; wherein X₁₂₄ is absent or present, and when X₁₂₄ is present, X₁₂₄=C or G; wherein X₁₂₅ is absent or present, and when X₁₂₅ is present, X₁₂₅=C or T; wherein X₁₂₆ is absent or present, and when X₁₂₆ is present, X₁₂₆=T, G or C; wherein X₁₂₇ is absent or present, and when X₁₂₇ is present, X₁₂₇=G, C or T; wherein X₁₂₈ is absent or present, and when X₁₂₈ is present, X₁₂₈=G; wherein X₁₂₉ is absent or present, and when X₁₂₉ is present, X₁₂₉=T; wherein X₁₃₀ is absent or present, and when X₁₃₀ is present, X₁₃₀=C; wherein X₁₃₁ is absent or present, and when X₁₃₁ is present, X₁₃₁=A; wherein X₁₃₂ is absent or present, and when X₁₃₂ is present, X₁₃₂=T; wherein X₁₃₃ is absent or present, and when X₁₃₃ is present, X₁₃₃=C; wherein X₁₃₄ is absent or present, and when X₁₃₄ is present, X₁₃₄=A; wherein X₁₃₅ is absent or present, and when X₁₃₅ is present, X₁₃₅=A; wherein X₁₃₆ is absent or present, and when X₁₃₆ is present, X₁₃₆=T; wherein X₁₃₇ is absent or present, and when X₁₃₇ is present, X₁₃₇=C; wherein X₁₃₈ is absent or present, and when X₁₃₈ is present, X₁₃₈=T; wherein X₁₃₉ is absent or present, and when X₁₃₉ is present, X₁₃₉=C; wherein X₁₄₀ is absent or present, and when X₁₄₀ is present, X₁₄₀=A; wherein X₁₄₁ is absent or present, and when X₁₄₁ is present, X₁₄₁=C; wherein X₁₄₂ is absent or present, and when X₁₄₂ is present, X₁₄₂=G; wherein X₁₄₃ is absent or present, and when X₁₄₃ is present, X₁₄₃=T; wherein X₁₄₄ is absent or present, and when X₁₄₄ is present, X₁₄₄=C; wherein X₁₄₅ is absent or present, and when X₁₄₅ is present, X₁₄₅=C; wherein X₁₄₆ is absent or present, and when X₁₄₆ is present, X₁₄₆=A, G, C or T; wherein X₁₄₇ is absent or present, and when X₁₄₇ is present, X₁₄₇=A, T, C or G; wherein X₁₄₈ is absent or present, and when X₁₄₈ is present, X₁₄₈=T, A, C or G; wherein X₁₄₉ is absent or present, and when X₁₄₉ is present, X₁₄₉=G, C, or A; wherein X₁₅₀ is absent or present, and when X₁₅₀ is present, X₁₅₀=G, C, A or T; wherein X₁₅₁ is absent or present, and when X₁₅₁ is present, X₁₅₁=A, C or T; wherein X₁₅₂ is absent or present, and when X₁₅₂ is present, X₁₅₂=C, G or T; wherein X₁₅₃ is absent or present, and when X₁₅₃ is present, X₁₅₃=T or C; wherein X₁₅₄ is absent or present, and when X₁₅₄ is present, X₁₅₄=A, T, G or C; wherein X₁₅₅ is absent or present, and when X₁₅₅ is present, X₁₅₅=C or T; wherein X₁₅₆ is absent or present, and when X₁₅₅ is present, X₁₅₆=G, T, A or C; wherein X₁₅₇ is absent or present, and when X₁₅₇ is present, X₁₅₇=A or G; wherein X₁₅₈ is absent or present, and when X₁₅₈ is present, X₁₅₈=C or T; wherein X₁₅₉ is absent or present, and when X₁₅₉ is present, X₁₅₉=A, T or G; wherein X₁₆₀ is absent or present, and when X₁₆₀ is present, X₁₆₀=T, A, C or G; wherein X₁₆₁ is absent or present, and when X₁₆₁ is present, X₁₆₁=A or T; wherein X₁₆₂ is absent or present, and when X₁₆₂ is present, X₁₆₂=G, T or A; wherein X₁₆₃ is absent or present, and when X₁₆₃ is present, X₁₆₃=A; wherein X₁₆₄ is absent or present, and when X₁₆₄ is present, X₁₆₄=C; wherein X₁₆₅ is absent or present, and when X₁₆₅ is present, X₁₆₅=G or A; wherein X₁₆₆ is absent or present, and when X₁₆₆ is present, X₁₆₆=C; wherein X₁₆₇ is absent or present, and when X₁₆₇ is present, X₁₆₇=T; wherein X₁₆₈ is absent or present, and when X₁₆₈ is present, X₁₆₈=C; wherein X₁₆₉ is absent or present, and when X₁₆₉ is present, X₁₆₉=G or A; wherein X₁₇₀ is absent or present, and when X₁₇₀ is present, X₁₇₀=T, G or A; wherein X₁₇₁ is absent or present, and when X₁₇₁ is present, X₁₇₁=C or T; wherein X₁₇₂ is absent or present, and when X₁₇₂ is present, X₁₇₂=T or C; wherein X₁₇₃ is absent or present, and when X₁₇₃ is present, X₁₇₃=A, G or T; wherein X₁₇₄ is absent or present, and when X₁₇₄ is present, X₁₇₄=G, A or T; wherein X₁₇₅ is absent or present, and when X₁₇₅ is present, X₁₇₅=T, C or A; wherein X₁₇₆ is absent or present, and when X₁₇₆ is present, X₁₇₆=C or T; wherein X₁₇₇ is absent or present, and when X₁₇₇ is present, X₁₇₇=C, A or T; wherein X₁₇₈ is absent or present, and when X₁₇₈ is present, X₁₇₈=G, A or C; wherein X₁₇₉ is absent or present, and when X₁₇₉ is present, X₁₇₉=C, A or T; wherein X₁₈₀ is absent or present, and when X₁₈₀ is present, X₁₈₀=C or T; wherein X₁₈₁ is absent or present, and when X₁₈₁ is present, X₁₈₁=A, C, G or T; wherein X₁₈₂ is absent or present, and when X₁₈₂ is present, X₁₈₂=A, T or C; wherein X₁₈₃ is absent or present, and when X₁₈₃ is present, X₁₈₃=G, T, A, or C; wherein X₁₈₄ is absent or present, and when X₁₈₄ is present, X₁₈₄=G, C or A; wherein X₁₈₅ is absent or present, and when X₁₈₅ is present, X₁₈₅=C or T; wherein X₁₈₆ is absent or present, and when X₁₈₆ is present, X₁₈₆=C, T or A; wherein X₁₈₇ is absent or present, and when X₁₈₇ is present, X₁₈₇=A or G; wherein X₁₈₈ is absent or present, and when X₁₈₈ is present, X₁₈₈=C or T; wherein X₁₈₉ is absent or present, and when X₁₈₉ is present, X₁₈₉=C, A or T; wherein X₁₉₀ is absent or present, and when X₁₉₀ is present, X₁₉₀=A, T, A or G; wherein X₁₉₁ is absent or present, and when X₁₉₁ is present, X₁₉₁=T, G, or A; wherein X₁₉₂ is absent or present, and when X₁₉₂ is present, X₁₉₂=A or T; wherein X₁₉₃ is absent or present, and when X₁₉₃ is present, X₁₉₃=T, or A; wherein X₁₉₄ is absent or present, and when X₁₉₄ is present, X₁₉₄=A or G; wherein X₁₉₅ is absent or present, and when X₁₉₅ is present, X₁₉₅=G, A, or T; wherein X₁₉₆ is absent or present, and when X₁₉₆ is present, X₁₉₆=G or T; wherein X₁₉₇ is absent or present, and when X₁₉₇ is present, X₁₉₇=T, C, or A; wherein X₁₉₈ is absent or present, and when X₁₉₈ is present, X₁₉₈=A or G; wherein X₁₉₉ is absent or present, and when X₁₉₉ is present, X₁₉₉=T or C; wherein X₂₀₀ is absent or present, and when X₂₀₀ is present, X₂₀₀=G, T, A or C; wherein X₂₀₁ is absent or present, and when X₂₀₁ is present, X₂₀₁=G, T or A; wherein X₂₀₂ is absent or present, and when X₂₀₂ is present, X₂₀₂=G, C or A; wherein X₂₀₃ is absent or present, and when X₂₀₃ is present, X₂₀₃=C or T; wherein X₂₀₄ is absent or present, and when X₂₀₄ is present, X₂₀₄=A or T; wherein X₂₀₅ is absent or present, and when X₂₀₅ is present, X₂₀₅=A, C or G; wherein X₂₀₆ is absent or present, and when X₂₀₆ is present, X₂₀₆=C or A; wherein X₂₀₇ is absent or present, and when X₂₀₇ is present, X₂₀₇=A or G; wherein X₂₀₈ is absent or present, and when X₂₀₈ is present, X₂₀₈=C; wherein X₂₀₉ is absent or present, and when X₂₀₉ is present, X₂₀₉=C; wherein X₂₁₀ is absent or present, and when X₂₁₀ is present, X₂₁₀=C or T; wherein X₂₁₁ is absent or present, and when X₂₁₁ is present, X₂₁₁=A or G; wherein X₂₁₂ is absent or present, and when X₂₁₂ is present, X₂₁₂=C or A; wherein X₂₁₃ is absent or present, and when X₂₁₃ is present, X₂₁₃=C or A; wherein X₂₁₄ is absent or present, and when X₂₁₄ is present, X₂₁₄=G; wherein X₂₁₅ is absent or present, and when X₂₁₅ is present, X₂₁₅=A; wherein X₂₁₆ is absent or present, and when X₂₁₆ is present, X₂₁₆=G; wherein X₂₁₇ is absent or present, and when X₂₁₇ is present, X₂₁₇=C; wherein X₂₁₈ is absent or present, and when X₂₁₈ is present, X₂₁₈=G; wherein X₂₁₉ is absent or present, and when X₂₁₉ is present, X₂₁₉=C; wherein X₂₂₀ is absent or present, and when X₂₂₀ is present, X₂₂₀=T; wherein X₂₂₁ is absent or present, and when X₂₂₁ is present, X₂₂₁=T; wherein X₂₂₂ is absent or present, and when X₂₂₂ is present, X₂₂₂=C; wherein X₂₂₃ is absent or present, and when X₂₂₃ is present, X₂₂₃=G; wherein X₂₂₄ is absent or present, and when X₂₂₄ is present, X₂₂₄=T; wherein X₂₂₅ is absent or present, and when X₂₂₅ is present, X₂₂₅=C; wherein X₂₂₆ is absent or present, and when X₂₂₆ is present, X₂₂₆=G; wherein X₂₂₇ is absent or present, and when X₂₂₇ is present, X₂₂₇=A; wherein X₂₂₈ is absent or present, and when X₂₂₈ is present, X₂₂₈=G; wherein X₂₂₉ is absent or present, and when X₂₂₉ is present, X₂₂₉=G; wherein X₂₃₀ is absent or present, and when X₂₃₀ is present, X₂₃₀=C; wherein X₂₃₁ is absent or present, and when X₂₃₁ is present, X₂₃₁=C; wherein X₂₃₂ is absent or present, and when X₂₃₂ is present, X₂₃₂=A; wherein X₂₃₃ is absent or present, and when X₂₃₃ is present, X₂₃₃=C; wherein X₂₃₄ is absent or present, and when X₂₃₄ is present, X₂₃₄=C.

Non-limiting exemplary sequences of mutant VEE virus subgenomic promoter region (−99/+30) are set forth in SEQ ID NOs: 2 to 74 (Kozak sequence in boldface if present; mutations in each mutant subgenomic promoter compared to wild type subgenomic promoter are annotated in FIG. 2). The sequences for SEQ ID NOs: 2, 14, 23, 41, 42, 45, 51, and 57 are not shown below because they are the same as the wild type subgenomic promoter (SEQ ID NO: 1).

(SEQ ID NO: 3) GACTTCCATCATATTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 4) GACTTGATGTCGACATCTTTGGTCAGAATCACCATATCGGCGCTCGCCAC ATCCTCCGCAGTCAGTTCATTTTCCAGACCGATTGACCCCTGGGTTTCTA CTTTTACTTCCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCTCTGCT GCCATTTAGGTGTGGGCAACACC (SEQ ID NO: 5) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTGAAAT CATTCAGCGACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGTA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 6) GACTCACCCACCACCACAGCAACGCGAGCACTCCATTTATCAGCGCAGGC AAACTGTTTCTAAAAGTTGCCGCCACCGTTGTTGGTCATCAATTTCACGC CCGGTAGTTCATCACGCAGACGCTCGGCTATTGCTATAGCCGCAGATAGT GTATCAGCACC (SEQ ID NO: 7) GACTTGATGCCGATATCTATGACCAGAATCGCCATATCAGCGCTCGCCTT ATCTTCCGCAGTCAGTTCATTTTCCGGACCAATTAATTCCTGGGTTTCCA CCTTCACTTCCCAGCCTTTCGCTTTCGCTGCAATATCCAGCGCCTCTGCT GCCATATAGGTATGGGCAACACC (SEQ ID NO: 8) GACTTGATGCCGATATCTTTGGTCAGATTCACCATATCAGCGCTCGCCAC TTCTTCCGCAGTCAGTTCATTTTCCAGATCAAATGACCCCTGGGCTTCTA CTTTCACTTCCCAGCCTTTCGCTTTCGCGGCAGTTTCCAGCGCCTCTGCT GCCATATAGGTATGGGCAACACC (SEQ ID NO: 9) GACTTCCATCATAGTTATGGCCATGACTACTCTAGATAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGGCCCCTATAACTCTCTACGGCTAACCTTA TTGGACTACGACATAGTCTAGCTCGCCAAGGCCACC (SEQ ID NO: 10) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTATCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACAACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 11) GACTCATCCAGCACCACAGCAACGCGGGTACCCCATTTATCAGCACGGGC GAACTGTTTCTTGAAGTTGCCACCGCCAAGGTTGGTCATCAATTTCACGC CCGGTAACTCATCACGCAGACACTCAGCTAATGCCATAGCCGCAGATTGT GTATCAGCACC (SEQ ID NO: 12) GACTTGATGCCGATATCTTTGATCAGTATCACCTTATCAACGCTAGCCAC AACTTCCGCAGTCAGTGATTTTTCCAGACCAATTGACCCCAGGGTTTCTA CTTTCACTTCCCTGCCTTTCGCATTCCCGGCACTTTCCAGCGCCTCTACT GCAATATAGGTATGGGCAACACC (SEQ ID NO: 13) GACTTCCATCATAGATATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CTTTCAGCTACCTGAGAGGGCCCCCATAACTCTCTACGGATAACCTGAAT GGACTACGACATAGTCTGGTCCGCCAAGGCCACC (SEQ ID NO: 15) GACTTGATGCCGATATCTTTGGTCAGAATCACCATATCAGCGCTCGCCAC ATCTTCCTGTAGTCAGTTCATTTCCCTGACCATTTGACCCCTGGGTTTCT ATTCTCACTTCCCAGCCTTTCGCTTTTGCAGCACTTTCTGGCGCCTCTGC AGCCGTATAGGTATGGGCAACACC (SEQ ID NO: 16) GACTTCCATCATAGTTATGGCCGTGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTAAAACTCTCTACCGCTAACCTGAA TGGACTACTACATAGTCTAATCCGCCAAGGCCACC (SEQ ID NO: 17) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCAGCCAAGGCCACC (SEQ ID NO: 18) GACTTCCATCATAGTTACGGCCATGACTCCTCAAGCAAGCAGTGTTAGAT CATTCAGCTACCTGAGAGGGACCCTATAACTCTCTACGGCTAACCTGAAT GGTCTTCGACATAGTCTAGACCGCCGAGGCCACC (SEQ ID NO: 19) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 20) GACTTGATGCCGTTATCTTTGGTCAGAATCACCATATCAGCGTTTGCCTC ATCTCCCGCAGTCAGTTCATTTTCCAGACCAATTGACCCCTGGGTTTCTA CTTTCACTTCCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCTCTGCT GCCATATTGGTATGGGCAACACC (SEQ ID NO: 21) GACTTCCATCATAGTTATGGCCATGACTATTCTAGCTAGCAGTGATAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGCCCGCCAAGGCCACC (SEQ ID NO: 22) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACAGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 24) GACTTGATGCCGATATCATTAGTCAAAATCACCAAATCAGCGCTCGCCAC ATCTTCCGCAGTCAGATCATTTTCCAGACCAATTGACCCCTGGGTTTCTA CTTTCACTTCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCTTCTGCTG CCATATAGGTATGGGCAGCACC (SEQ ID NO: 25) GACTCACCCAGCATCACAGCATCGCGGGCCCCCCATTTATCAGCACGGTA AACAGTTTCTTAAAGCTGTCGCCGCCGTGGTTGGTCATCAATTCCACGCC CGGTAATTCGTCATGCAGACGCTCATCTAATGCCATAGCCGCTGATTGTG TATCAGCACC (SEQ ID NO: 26) GACTTGATGTCGATATCTTTGGTCAGAATCACCATATCAGCGCTCGCCAC ATCTTCCGCAATCAGTTCATTTTCCAGACCAATTGATGCCTGGGTTTCTA CTTTCACTTCCCAGCCTTTCGCTTTCGCGGCACTTTTCAGCGCCTCTGCT GCCATATAGGTATGGGCAACACC (SEQ ID NO: 27) GACTTCCATCATAGTTATGGCCATGACTACTCTACCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 28) GACTTCAATCATAGTCATGGCTATGACTACTCCAGCTAGCAGTGTTAAAT CATTCAGCTACCTGGGAGTGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTTCGCCAAGGCCACC (SEQ ID NO: 29) GACTTCCATCATAGTTATGACCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGCCCGCCAAGGCCACC (SEQ ID NO: 30) GACTTCCATCATAGTTTTGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTTTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 31) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTTTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 32) GACTTCCATCTTAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCTTATAACTCTCTACTGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 33) GACTTGATGCCGATATCTTTGGCAGAATCACCATATCAGCGCTCGCACAT CTTCCGCAGTCAGTTCATTATCCAGACTGATTGACCCCTGGGTTTCTACT TTCACTTCCCAGCCTTTCGCCTTCGCGGCACTTTCCAGCGCCTCTGCTGC CATATAGGTATGGGCAACACC (SEQ ID NO: 34) GACTTCCATCATAGTTACGGCCATGACTACTCGAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGACCTTTTAACTCTCTACGGCTAACCTGTA TGGACTACGACAAAGTCTAGTCAGCCAAGGCCACC (SEQ ID NO: 35) GACTTGATGCCGATATCTTTGGCAGAATCACCATATCAGCGCTCGCACAT CTTCCGCAGTCAGTTCATTATCCAGACTGATTGACCCCTGGGTTTCTACT TTCACTTCCCAGCCTTTCGCCTTCGCGGCACTTTCCAGCGCCTCTGCTGC CATATAGGTATGGGCAACACC (SEQ ID NO: 36) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAGAT CATTCAGCCACCTGAGAGGGGCCCCTAAAACTCTCTACGGCTGACCTGAA TGGACTACGACATAGTTTAGTTCACCAAGGCCACC (SEQ ID NO: 37) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGACCCCTATGACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCAGCCAAGGCCACC (SEQ ID NO: 38) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA GAGCGCTTCCTCAGGCCACC (SEQ ID NO: 39) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGATGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 40) GACTTCCATCATCGTTATGGCCATGGCTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGTCTAACCGGAA TGGACTACGACATAGTCTAGTCCGCCAAGCCCACC (SEQ ID NO: 43) GACTTGATGCCGATATCTTTGGTCAGAATCACCATATCAGCGCTCGCCAC ATCTTCCGCAGTCAGTTCATTTTCTAGACCAATTGACCCCTGGGTTTCTA CTTTCACTTCCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCTCTGCT GCCATATAGGTATGGGCAACACC (SEQ ID NO: 44) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACTACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 46) GACTTGATGCCGATATCCTTGCTCAGGATCACCGTATCAGCGCTCGCCAC ATCTTCGGCAGTCAGTTCATTTTCCATACCAACTGGCCCCTGGGTTTCTA CTTTCGCGTCCCAGCCTTTCGCTTTCGTGGCACTTTCCAGCATCTCTGCT GCCATATAGGTATGTGCAACACC (SEQ ID NO: 47) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGTAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 48) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTGAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA CGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 49) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTAAATC ATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAAT GCACTACGACATAGTCTAGTCCGCACAGGCCACC (SEQ ID NO: 50) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGGA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 52) GACTTGACGCCGACATCTATGGTCAGAACCACCAAATCAGTGCTCGCCTC ATCTTCTGCAGTAAGTTCATTTTCCAGACCAATCGACCCCTGGGTTTCTT CTTTCTCTTCCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCCCTGCT GCCATATAGGCATGGGTTACACC (SEQ ID NO: 53) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTATGGCTAACCTGAA TGGACTACGACTTAGTCTAGTCCGCCATGGCCACC (SEQ ID NO: 54) GACTTCCAGCATAGTTATCGCCAAGACTACTCTAGCTAGCATTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATTGTCTAGTCCGCCAAGGCCACCTGAAGAGCGCTTCCT CAGCCCACC (SEQ ID NO: 55) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAGCCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 56) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGGGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 58) GACTTGATGCAGGTATCTTTGGTCGGAACCACCATATCAACGCTCTCCAC ATCCTCCGCAGTCAGCACATTATCCAGACCAATTGACCCCTGTGTTTCTA CTATCTCTTCCCCACCTTTCGCTTTCGCGGCGCTTTCCTGCGCCTCTGCC GCCATATAGGTATGGGCAACACC (SEQ ID NO: 59) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGAGTTAATC ATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCAAACCTGAAT GGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 60) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTTCCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 61) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA AGGTCTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 62) GACTTCCATCATAGTTACGGCCATGACCACTATAGCTAGCAGTGTTAAAT CATTAAGCTACCTAAGAGGGGCCCCCTTAACTATATACGGCTAACCTGAA GGGCTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 63) GACTTCCATCATAGTTATGCCCTTGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 64) GACTCACCCAGCACCACAGCAACACGGGCTACCCATTCATCAGCCTGGGC AGACTGTTTCTTAAAGTTGCCGACGCCGCGCTTGGTCATCAATCTCACGT CCGGTAATTCATCACTCAGACGCTCTGCTAATGCCATAGCCGCAGATTGT GTATCAGCACC (SEQ ID NO: 65) GACTTGATGCCGATATCTATGGTCAGGATCACCATTACGTCGCTCGCCAC ATCTTTCGCAGTCAGTTCATTTTCCAGGCCTATTGACCCCTGGGTTTCTA TTATCACTTCCCATCCTTTCGCATTCGCGGTACTTTCCAGCGCCTCTGCC GCCAAATAGGTGTAGACTACACC (SEQ ID NO: 66) GACTTCCATCATAATTAAGGCCATGACTACTCAAGCTAGCAGTGTTAAAT CGTTCAGTCGCCTAAGAGGGGACACTTTAACTCTCTACGGCTAACCTGTA TCGACTACGACTTAGTCTAGTCTGCCAAGGCCACC (SEQ ID NO: 67) GACTTGATGCCGATATCTTCGGTCAGAATCACCATATCAGCGCTCACAAC ATCTTCACCAGTCAGTAGATTTTCCAGACCAATTGACCCGTGGGCTTCTA CTTTCACTTCCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCTCTGCT GCCTATAGGCATGGGCAACACC (SEQ ID NO: 68) GACTTGATGCCGATATCTCTGGTCAGAATCACCATATCAGCGCTCGCCAT ATCTTCCGCAGTCAGTTCATTTTCCAAACCAATTGACCCATGGGTTTCTA CCTTCACTTCCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCTCTGCT GCCATATAGTTATGGGCAACACC (SEQ ID NO: 69) GACTTCCATCATAGTTACGGCCACGTCTACTCTAGCTTGCAGTGTCAAAT CAATCAGCCACC (SEQ ID NO: 70) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAATGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGCA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 71) GACTTGATGACGATATCTTTGGTCGGAATCACCATATCAATGCTCGCCAC ATCTTCCGCGGTAAGTTCATCTTCCAGACAATTGACCCCTGGGTTACTAC TATCACTTCCCTGCCTTACGCTTTGCGGCACTTACCAGCGCCTCCATTGC CATATAGGAATGGGCAACACCTGAAGAGCGCTTCGTCGAGGCCACC (SEQ ID NO: 72) GACTTCCATCATAGATATGGCCAAGACTAGTCTAGCTATTAGTGATAAAT CATTCAACTACCTGAGAGGGGCCCCCATTACTCACTACGGCTAACCTGAA CGGAGTACGGCATTGTCTAGTCTGCCAAGGCCACC (SEQ ID NO: 73) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTGAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC (SEQ ID NO: 74) GACTTGATGCCGATATCTTAGGTCAGAATCACCATATCAGTGATCGCAAC ATCTTCCGCAGACAATTCATTTTCCAAACCAATTGACCCCTGGGTTTCTA CTTTCGCTTCTCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCTCTGCT GTCATAAAGGTATGGGCAACACC

A subgenomic promoter library, as used herein, refers to a plurality of subgenomic promoters, which includes mutant subgenomic promoters, and in some embodiments can include a wild type subgenomic promoter. In some instances, the subgenomic promoters in the library, each comprises a nucleic acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to nucleic acid sequences of SEQ IDs NO: 1-74 and 81. In some embodiments, the subgenomic promoters in the library, each comprises a nucleic acid sequence at least 80% identical to nucleic acid sequences of SEQ ID NO: 1-74 and 81. For example, an engineered subgenomic promoter may comprise a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the nucleotide sequence identified by any one of SEQ ID NOs: 1-74 and 81, and is able to regulate the expression (e.g., activate or repress) the sequence to which it is operably linked. In some embodiments, an engineered subgenomic promoter comprises a nucleotide sequence that is 95-99% identical to the nucleotide sequence identified by any one of SEQ ID NOs: 1-74 and 81, and is able to regulate the expression (e.g., activate or repress) the sequence to which it is operably linked. In some embodiments, an engineered subgenomic comprises a nucleotide sequence that is 95%-99%, 95%-98%, 95%-97%, 95%-96%, 96%-99%, 96%-98%, 96%-97%, 97%-99%, 97%-98%, or 98%-99% identical to the nucleotide sequence identified by any one of SEQ ID NOs: 1-74 and 81, and is able to regulate the expression (e.g., activate or repress) the sequence to which it is operably linked.

In some embodiments, each of the subgenomic promoters in the library is flanked by a pair of restriction endonucleases site at the 5′ and the 3′ end. A restriction endonuclease refers to an enzyme that cleaves DNA into fragments at or near specific recognition sites within the molecule known as restriction sites. Restrictions enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix. These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction digestion; meanwhile, host DNA is protected by a modification enzyme (a methyltransferase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system. Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially. These enzymes are routinely used for DNA modification in laboratories, and they are a vital tool in molecular cloning.

Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. The recognition sequences can also be classified by the number of bases in its recognition site, usually between 4 and 8 bases, and the number of bases in the sequence will determine how often the site will appear by chance in any given genome. Non-limiting examples of restriction endonucleases sites include DNA sequences that are recognized by SapI, AtaII, Acc65I, ApaI, BamHI, BcII, BgIII, ClaI, EcoRI, EcoRV, HindIII, KasI, KpnI, Mfel, Mlul, NspI, PciI, PstI, SacI, SphI, XbaI, or XhoI.

Other aspects of the present disclosure provide engineered subgenomic promoters having differential activities. “Having differential activities” means the activity of an engineered subgenomic promoter is higher or lower compared to each other. In some embodiments, the activity of an engineered subgenomic promoter is different from (higher or lower) the activity of another engineered subgenomic promoter by at least 10% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, or 1000-fold). In some embodiments, the activity of an engineered subgenomic promoter is different from (higher or lower) the activity of another subgenomic promoter by 10%-100%. For example, the activity of an engineered subgenomic promoter may be different from (higher or lower) the activity of another engineered subgenomic promoter by 10%-100%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 30%-40%, 40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100%. For example, the activity of an engineered subgenomic promoter may be different from (higher or lower) the activity of another engineered subgenomic promoter by 1-1000 fold. For example, the activity of an engineered subgenomic promoter may be different from (higher or lower) the activity of another engineered subgenomic promoter by 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 5-1000, 5-900, 5-800, 5-700, 5-600, 5-500, 5-400, 5-300, 5-200, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 5-9, 5-8, 5-7, 5-6, 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-1000, 20-900, 20-800, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-1000, 30-900, 30-800, 30-700, 30-600, 30-500, 30-400, 30-300, 30-200, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-1000, 40-900, 40-800, 40-700, 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 50-90, 50-80, 50-70, 50-60, 60-1000, 60-900, 60-800, 60-700, 60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60-90, 60-80, 60-70, 70-1000, 70-900, 70-800, 70-700, 70-600, 70-500, 70-400, 70-300, 70-200, 70-100, 70-90, 70-80, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500, 80-400, 80-300, 80-200, 80-100, 80-90, 90-1000, 90-900, 90-800, 90-700, 90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-800, 600-700, 700-1000, 700-900, 700-800, 800-1000, 800-900, or 900-1000 fold. Methods of measuring the activities of a promoter (e.g., an engineered subgenomic promoter) are known to those skilled in the art, e.g., as described in Jeyaseelan et al., Nucleic Acids Research. 29 (12), 2001; Allard et al., Cell Notes (21), 2008; and Zaslaver et al., Nature Methods. 3 (8): 623-628, 2006, each of which is incorporated herein by reference.

II. Engineered Nucleic Acid, Expression Cassettes and Vectors (i) Engineered Nucleic Acid

Further provided herein are engineered nucleic acids (e.g., constructs) containing the engineered subgenomic promoters described herein. In some embodiments, an engineered subgenomic promoter is operably linked to a transgene (e.g., a nucleotide sequence encoding a gene). A promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and/or expression of that sequence.

In some embodiments, an engineered subgenomic promoter is operably linked to a nucleotide sequence encoding a transgene, such that activation of the engineered subgenomic promoter results in expression of the transgene. The signal of the transgene may be detected and its intensity is an indication of the level of activation of the subgenomic promoter. As such, by comparing the signal from the transgene, the activities of a subgenomic promoter can be compared. The engineered subgenomic promoter provided herein can be operably linked to a transgene encoding any molecule of interest, such as nucleic acids (e.g., mRNA, inhibitory nucleic acids, etc.), endogenous proteins, recombinant proteins, detectable proteins, therapeutic proteins, enzymes, growth factors, cytokines, etc.

In some embodiments, the transgene encodes a detectable molecule, such as a detectable protein. In some embodiments, a detectable protein is a fluorescent protein. A fluorescent protein is a protein that emits a fluorescent light when exposed to a light source at an appropriate wavelength (e.g., light in the blue or ultraviolet range). Suitable fluorescent proteins that may be used as a detectable protein in the sensor circuit of the present disclosure include, without limitation, eGFP, eYFP, eCFP, mKate2, mCherry, mPlum, mGrape2, mRaspberry, mGrape1, mStrawberry, mTangerine, mBanana, and mHoneydew. In some embodiments, a detectable protein is an enzyme that hydrolyzes a substrate to produce a detectable signal (e.g., a chemiluminescent signal). Such enzymes include, without limitation, beta-galactosidase (encoded by LacZ), horseradish peroxidase, or luciferase. In some embodiments, the detectable molecule is a fluorescent RNA. A fluorescent RNA is an RNA aptamer that emits a fluorescent light when bound to a fluorophore and exposed to a light source at an appropriate wavelength (e.g., light in the blue or ultraviolet range). Suitable fluorescent RNAs that may be used include, without limitation, Spinach and Broccoli (e.g., as described in Paige et al., Science Vol. 333, Issue 6042, pp. 642-646, 2011, incorporated herein by reference).

In some embodiments, an engineered subgenomic promoter is operably linked to a transgeneencoding a therapeutic molecule. A “therapeutic molecule” is a molecule that has therapeutic effects on a disease or condition, and may be used to treat a diseases or condition.

Therapeutic molecules of the present disclosure may be nucleic acid-based or protein or polypeptide-based. In some embodiments, the engineered subgenomic promoter drives the expression of the therapeutic molecule in a desired cell type (e.g., cancer cell) but not in other cell types, due to the engineered subgenomic promoter's cell-specific activity. As such, targeted therapy of diseases (e.g., cancer) is achieved.

In some embodiments, an engineered subgenomic promoter is operably linked to a transgene encoding a therapeutic nucleic acid. In some embodiments, nucleic acid-based therapeutic molecule may be an RNA interference (RNAi) molecule (e.g., a microRNA, siRNA, or shRNA) or a nucleic acid enzyme (e.g., a ribozyme). RNAi molecules and there use in silencing gene expression are familiar to those skilled in the art. In some embodiments, the RNAi molecule targets an oncogene. An oncogene is a gene that in certain circumstances can transform a cell into a tumor cell. An oncogene may be a gene encoding a growth factor or mitogen (e.g., c-Sis), a receptor tyrosine kinase (e.g., EGFR, PDGFR, VEGFR, or HER2/neu), a cytoplasmic tyrosine kinase (e.g., Src family kinases, Syk-ZAP-70 family kinases, or BTK family kinases), a cytoplasmic serine/threonine kinase or their regulatory subunits (e.g., Raf kinase or cyclin-dependent kinase), a regulatory GTPase (e.g., Ras), or a transcription factor (e.g., Myc). In some embodiments, the oligonucleotide targets Lipocalin (Lcn2) (e.g., a Lcn2 siRNA). One skilled in the art is familiar with genes that may be targeted for the treatment of cancer.

In some embodiments, an engineered subgenomic promoter is operably linked to a transgene encoding a therapeutic protein. Non-limiting examples of protein or polypeptide-based therapeutic molecules include enzymes, regulatory proteins (e.g., immuno-regulatory proteins), antigens, antibodies or antibody fragments, and structural proteins.

Suitable enzymes (for operably linking to a subgenomic promoter) for some embodiments of this disclosure include, for example, oxidoreductases, transferases, polymerases, hydrolases, lyases, synthases, isomerases, and ligases, digestive enzymes (e.g., proteases, lipases, carbohydrases, and nucleases). In some embodiments, the enzyme is selected from the group consisting of lactase, beta-galactosidase, a pancreatic enzyme, an oil-degrading enzyme, mucinase, cellulase, isomaltase, alginase, digestive lipases (e.g., lingual lipase, pancreatic lipase, phospholipase), amylases, cellulases, lysozyme, proteases (e.g., pepsin, trypsin, chymotrypsin, carboxypeptidase, elastase), esterases (e.g. sterol esterase), disaccharidases (e.g., sucrase, lactase, beta-galactosidase, maltase, isomaltase), DNases, and RNases.

A regulatory protein may be, in some embodiments, a transcription factor or an immunoregulatory protein. Non-limiting, exemplary transcriptional factors include: those of the NFkB family, such as Rel-A, c-Rel, Rel-B, p50 and p52; those of the AP-1 family, such as Fos, FosB, Fra-1, Fra-2, Jun, JunB and JunD; ATF; CREB; STAT-1, -2, -3, -4, -5 and -6; NFAT-1, -2 and -4; MAF; Thyroid Factor; IRF; Oct-1 and -2; NF-Y; Egr-1; and USF-43, EGR1, Sp1, and E2F1. Other transcription factors may be operably linked to a subgenomic promoter, as provided herein. As used herein, an immunoregulatory protein is a protein that regulates an immune response. Non-limiting examples of immunoregulatory proteins include: antigens, adjuvants (e.g., flagellin, muramyl dipeptide), cytokines including interleukins (e.g., IL-2, IL-7, IL-15 or superagonist/mutant forms of these cytokines), IL-12, IFN-gamma, IFN-alpha, GM-CSF, FLT3-ligand), and immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3, or single chain/antibody fragments of these molecules). Other immunoregulatory proteins may be operably linked to a subgenomic promoter, as provided herein.

In some embodiments, the transgene encodes a heavy chain and/or a light chain of an antibody. In some embodiments, one of the subgenomic promoters described herein is operably linked to a transgene encoding the heavy chain of an antibody. In some embodiments, one of the subgenomic promoters described herein is operably linked to a transgene encoding the light chain of an antibody. An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target antigen, through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, NANOBODIES®, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins are assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The term “isolated antibody” used herein refers to an antibody substantially free from naturally associated molecules, i.e., the naturally associated molecules constituting at most 20% by dry weight of a preparation containing the antibody. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, and HPLC.

A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Non-limiting examples of antibodies and fragments thereof include: an anti-CTLA-4 antibody such as ipilimumab (YERVOY®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), alemtuzumab (CAMPATH®), indicated for B cell chronic lymphocytic leukemia), gemtuzumab (MYLOTARG, hP67.6, anti-CD33, indicated for leukemia such as acute myeloid leukemia), rituximab (RITUXAN®), tositumomab (BEXXAR®, anti-CD20, indicated for B cell malignancy), MDX-210 (bispecific antibody that binds simultaneously to HER-2/neu oncogene protein product and type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI)), oregovomab (OVAREX®, indicated for ovarian cancer), edrecolomab (PANOREX®), daclizumab (ZENAPAX®), palivizumab (SYNAGIS®, indicated for respiratory conditions such as RSV infection), ibritumomab tiuxetan (ZEVALIN®, indicated for Non-Hodgkin's lymphoma), cetuximab (ERBITUX®), MDX-447, MDX-22, MDX-220 (anti-TAG-72), IOR-C5, IOR-T6 (anti-CD1), IOR EGF/R3, celogovab (ONCOSCINT® OV103), epratuzumab (LYMPHOCIDE®), pemtumomab (THERAGYN®), Gliomab-H (indicated for brain cancer, melanoma). In some embodiments, the engineered nucleic acid comprises a subgenomic promoter selected from the subgenomic promoter library and a heavy chain or a light chain of an anti-CTLA-4 antibody coding sequence operably linked to the subgemomic promoter. In some embodiments, the anti-CTLA-4 antibody is ipilimumab.

(ii) Expression Cassettes and Vectors

The present disclosure also provides expression cassettes comprising one or more engineered nucleic acids described herein. An expression cassettes, as used herein, refers to a DNA fragment comprises one or more engineered nucleic acid described herein, for expression of one or more transgenes. In some embodiments, the expression cassette comprises one engineered nucleic acid, which comprises a first subgenomic promoter selected from the subgenomic promoter library and a first transgene. Alternatively or in addition, the expression cassette can comprise a second engineered nucleic acid, which comprises a second subgenomic promoter selected from the subgenomic promoter library and a second transgene. In other embodiments, the expression cassette can comprise 1, 2, 3, 4, 5, 6, 7, 9, 10, or more engineered nucleic acids described herein. The one or more subgenomic promoter selected from the subgenomic promoter library can be the same or different. The one or more transgenes can be the same or different. In some instances, the expression cassette is an antibody expression cassette. An antibody expression cassette, as used herein, refers to a DNA fragment that expresses both the heavy chain and the light chain of the antibody to generate a functional antibody. In some examples, the antibody expression cassette comprises a first engineered nucleic acid expressing the heavy chain of an antibody; and a second engineered nucleic acid expressing the light chain of an antibody. In some embodiments, the antibody expression cassette comprises a first engineered nucleic acid comprising a first engineered subgenomic promoter selected from the engineered subgenomic promoter library operably linked to a first transgene, and the first transgene encodes a heavy chain of an antibody; and a second engineered nucleic acid comprising a second engineered subgenomic promoter selected from the engineered subgenomic promoter library operably linked to a second transgene, and the second transgene encodes a light chain of an antibody. In some embodiments, the first subgenomic promoter driving the expression of the heavy chain of the antibody, and the second subgenomic promoter driving the expression of the light chain of the same antibody are selected from the subgenomic promoter library to ensure the optimal production of functional antibodies.

Further provided herein are vectors that comprise one or more engineered nucleic acids or one or more expression cassettes described in the present disclosure. A vector includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term vector includes cloning and expression vehicles, as well as viral vectors. In some instances, the vector is a plasmid, RNA replicon, linear double stranded DNA, viral vector, liposome or nanoparticle. As used herein, the term “RNA replicon” refers to a self-replicating genetic element comprised a RNA that replicates from one origin of replication. In some embodiments, the self-amplifying replicon RNA is derived from an alphavirus. Distinct from host mRNA, alphavirus replicon RNAs encode a set of four nonstructural proteins (nsPs 1-4) that are responsible both for genome replication and, when engineered to include genes encoding non-virus products, such as a transgene, provide for transcription of such non-viral products under the subgenomic promoter.

Cells comprising the engineered nucleic acids, expression cassettes and vectors are within the scope of the disclosure. The cell can be any cell suitable for producing the transgene. In some embodiments, the cells are mammalian cells, plant cells, insect cells, bacterial cells or fungi cells. In some examples, the cells are mammalian cells.

III. Applications of Subgenomic Promoter Library

(i) Production of Transgene Encoded Molecules

The present disclosure, at least in part, relates to a method of producing one or more transgene encoded molecules using the engineered nucleic acid described herein.

In some embodiments, the method comprises constructing one or more engineered nucleic acids described herein, for expression of one or more transgenes. In some examples, the engineered nucleic acid is prepared by recombinant technology as exemplified below. One or more transgenes are operably linked to a subgenomic promoter selected from the subgenomic promoter library provided herein and cloned into an expression vector.

In some embodiments, one or more engineered nucleic acids or one or more vectors (e.g., expression vectors) including engineered nucleic acids encoding the one or more transgenes may be introduced into suitable host cells for producing the transgene encoded molecules. The engineered nucleic acids or vectors can be introduced into host cells by conventional methods, e.g., liposome transfection, electroporation, or calcium phosphate precipitation. The host cells are cultured under suitable conditions for expression of the transgene thereof. In other embodiments, one or more engineered nucleic acid or one or more vectors including engineered nucleic acids encoding the one or more transgenes may be introduced into a host animal for producing the transgene encoded molecules in vivo by conventional methods in the art. Non-limiting exemplary suitable host animals can be humans or non-human mammals such as mice, goats, rabbits, pigs, donkeys, cows, or camels. Methods of introducing an engineered nucleic acid or vectors into a host animal are conventional in the art, for example, by recombinant viruses such as recombinant adeno-associated virus, recombinant lentivirus, liposome, nanoparticles, or microinjection.

Such transgene encoded molecules thereof can be recovered from the cultured cells (e.g., from the cells or the culture supernatant) via a conventional method, e.g., affinity purification.

Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium.

Non-limiting examples transgene encoded molecules include therapeutic molecules (e.g., cytokines, peptide based agonists), detectable molecules (e.g., fluorescent proteins), antibodies or antibody fragments, siRNA, enzymes, regulatory proteins (e.g., immuno-regulatory proteins), antigens, and structural proteins.

(ii) Fine Tune Expression Level of Multiple Transgenes on One Replicon RNA

Also provided herein are methods for selecting and using subgenomic promoters with differential levels to fine tune expression level of multiple transgenes on one vector. The methods comprise constructing an expression cassette library. In some embodiments, each expression cassette comprises more than one engineered nucleic acids. Each engineered nucleic acid can comprise a subgenomic promoter selected from the subgenomic promoter library operably linked to a different transgene. In some embodiments, the expression cassette is an antibody cassette. In some embodiments, the transgene is the heavy chain and the light chain of the antibody. In other embodiments, the transgene encodes a therapeutic molecule or a detectable molecule.

The library of the constructed expression cassettes can be introduced into suitable host cell. The host cells are cultured under suitable conditions for expression of the transgene thereof. The cells expressing desired level of each transgene can be selected by conventional methods (e.g., FACS sorting). In some embodiments, the transgenes encodes for a heavy chain and a light chain of an antibody, and the cells that express a desired level of functional antibody can be selected. In some embodiments, the nucleic acid sequences of the subgenomic promoters driving the transgenes can then be determined (e.g., by nucleic acid sequencing). Such subgenomic promoters can be selected to produce the transgenes at desired level. In some examples, the pair of subgenomic promoters that produce optimal level of functional antibodies can be selected for production of the antibody.

IV. General Techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995). Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES Example 1: Construction of Alphavirus Replicon Subgenomic Promoter Library

To precisely tune expression level of multiple transgenes from a single alphavirus replicon, libraries of subgenomic promoter (SGP) with differential activity (increased or decreased relative another subgenomic promoter in the library or wild type) was constructed. Subgenomic promoter mutants were generated using error-prone PCR, and specific mutants with varying expression level were screened. Utilization of the subgenomic promoter library is demonstrated by optimization of antibody expression.

Alphavirus derived wild type subgenomic promoter (assuming position +1 is the transcription start site) sequence from position −99 to +30 was flanked by SapI restriction site and subsequently cloned into vector pBjh6018. The nucleic acid sequence of the wild type subgenomic promoter is set forth in SEQ ID NO: 1 (−99 to 0 underlined; Kozak sequence in bold face):

(SEQ ID NO: 1) GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAAT CATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAA TGGACTACGACATAGTCTAGTCCGCCAAGGCCACC

The sequence of SapI flanked wild type subgenomic promoter is set forth in SEQ ID NO: 76 (SapI site in boldface):

(SEQ ID NO: 76) GCTCTTCAGACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCA GTGTTAAATCATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGG CTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGGCCACCTGAA GAGC

Mutants of the subgenomic promoter were created by error prone PCR using the GeneMorphII Random mutagenesis Kit (Agilent). The primers used in the PCR are:

Forward primer: (SEQ ID NO: 77) TATTGGGCGCTCTTCAGACT Reverse primer: (SEQ ID NO: 78) GAGGAAGCGCTCTTCAGGTG

The PCR products contained various mutations of the subgenomic promoter, which were subjected to further screening for mutant subgenomic promoters that have differential activity in driving downstream gene expression.

The PCR products containing mutant subgenomic promoters (mutant SGP) were digested by SapI (SapI-mutant SGP-SapI) and ligated into the pBjh6031 vector, which comprises one wild type subgenomic promoter driving the expression of mVenus, and a Ptet promoter driving the expression of Red Fluorescence Protein (RFP), illustrated as Seq1_D1-CMV_nsP(1-4)-SGP1(−98/+30)_mVenus_3′UTR_SapI-Ptet-RFP-SapI_mKate_3′UTR-rb_glob_PA_Seq2. The sequence flanked by SapI sites are in Boldface. The SapI-mutant SGP-SapI was inserted into pBjh6031 to replace the SapI-Ptet-RFP-SapI. The resulting vectors comprises one wild type subgenomic promoter (referred to as SGP1) driving the expression of mVenus, and a mutant subgenomic promoter driving the expression of mKate, illustrated as Seq1_D1-CMV_nsP(1-4)-SGP1(−98/+30)_mVenus_3′UTR_SapI-mutant SGP-SapI_mKate_3′UTR-rb_glob_PA_Seq2. The ligated vectors were transformed into EC100D electrocompetent cell (Lucigen) by electroporation for further propagation and vector purification. The mutant SGP is referred to as SGP2.

One million of A549 cells were transfected with 7.5 g of purified vectors, cultured for proper expression of mVenus and mKate and analyzed by Fluorescence Activated Cell Sorting (FACS). Cells expressing various levels of mVenus and mKate were sorted into eight different populations according to the gating strategy illustrated in FIG. 1A. Total RNA of each cell population was isolated for cDNA generation. cDNAs were digested with SapI and inserted into pBjh6031 for propagation and subsequent sequencing of the SGP2 region. For each gated region in FIG. 1A, 12 clones were picked and SGP2 region of the vectors isolated from each clone were sequenced. The results of the sequencing are shown in Table 1 and the mutations in each mutant subgenomic promoter are annotated as compared to wild type subgenomic promoter in FIG. 2.

TABLE 1 SEQ ID ID −99/+30/GCCACC (−99 underlined; Kozak sequence in boldface) Length NO WT GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 1 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P4-1 Sequencing failed 0 P4-2 Sequencing failed 0 P4-3 Sequencing failed 0 P4-4 Sequencing failed 0 P4-5 Sequencing failed 0 P4-6 Sequencing failed 0 P4-7 Sequencing failed 0 P4-8 Sequencing failed 0 P4-9 Sequencing failed 0 P4-10 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 1 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P4-11 GACTTCCATCATATTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 3 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA ACTACGACATAGTCTAGTCCGCCAAGGCCACC P4-12 GACTTGATGTCGACATCTTTGGTCAGAATCACCATATCGGCGCTCGCCACAT 173 4 CCTCCGCAGTCAGTTCATTTTCCAGACCGATTGACCCCTGGGTTTCTACTTT TACTTCCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCTCTGCTGCCATT TAGGTGTGGGCAACACC P5-1 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTGAAATCA 135 5 TTCAGCGACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGTATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P5-2 GACTCACCCACCACCACAGCAACGCGAGCACTCCATTTATCAGCGCAGGCAA 161 6 ACTGTTTCTAAAAGTTGCCGCCACCGTTGTTGGTCATCAATTTCACGCCCGG TAGTTCATCACGCAGACGCTCGGCTATTGCTATAGCCGCAGATAGTGTATCA GCACC P5-3 GACTTGATGCCGATATCTATGACCAGAATCGCCATATCAGCGCTCGCCTTAT 173 7 CTTCCGCAGTCAGTTCATTTTCCGGACCAATTAATTCCTGGGTTTCCACCTT CACTTCCCAGCCTTTCGCTTTCGCTGCAATATCCAGCGCCTCTGCTGCCATA TAGGTATGGGCAACACC P5-4 GACTTGATGCCGATATCTTTGGTCAGATTCACCATATCAGCGCTCGCCACTT 173 8 CTTCCGCAGTCAGTTCATTTTCCAGATCAAATGACCCCTGGGCTTCTACTTT CACTTCCCAGCCTTTCGCTTTCGCGGCAGTTTCCAGCGCCTCTGCTGCCATA TAGGTATGGGCAACACC P5-5 GACTTCCATCATAGTTATGGCCATGACTACTCTAGATAGCAGTGTTAAATCA 136 9 TTCAGCTACCTGAGAGGGGGCCCCTATAACTCTCTACGGCTAACCTTATTGG ACTACGACATAGTCTAGCTCGCCAAGGCCACC P5-6 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTATCAGTGTTAAATCA 135 10 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACAACATAGTCTAGTCCGCCAAGGCCACC P5-7 Sequencing failed 0 P5-8 GACTCATCCAGCACCACAGCAACGCGGGTACCCCATTTATCAGCACGGGCGA 161 11 ACTGTTTCTTGAAGTTGCCACCGCCAAGGTTGGTCATCAATTTCACGCCCGG TAACTCATCACGCAGACACTCAGCTAATGCCATAGCCGCAGATTGTGTATCA GCACC P5-9 GACTTGATGCCGATATCTTTGATCAGTATCACCTTATCAACGCTAGCCACAA 173 12 CTTCCGCAGTCAGTGATTTTTCCAGACCAATTGACCCCAGGGTTTCTACTTT CACTTCCCTGCCTTTCGCATTCCCGGCACTTTCCAGCGCCTCTACTGCAATA TAGGTATGGGCAACACC P5-10 GACTTCCATCATAGATATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCT 134 13 TTCAGCTACCTGAGAGGGCCCCCATAACTCTCTACGGATAACCTGAATGGAC TACGACATAGTCTGGTCCGCCAAGGCCACC P5-11 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 1 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P5-12 GACTTGATGCCGATATCTTTGGTCAGAATCACCATATCAGCGCTCGCCACAT 174 15 CTTCCTGTAGTCAGTTCATTTCCCTGACCATTTGACCCCTGGGTTTCTATTC TCACTTCCCAGCCTTTCGCTTTTGCAGCACTTTCTGGCGCCTCTGCAGCCGT ATAGGTATGGGCAACACC P6-1 GACTTCCATCATAGTTATGGCCGTGACTACTCTAGCTAGCAGTGTTAAATCA 135 16 TTCAGCTACCTGAGAGGGGCCCCTAAAACTCTCTACCGCTAACCTGAATGGA CTACTACATAGTCTAATCCGCCAAGGCCACC P6-2 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 17 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCAGCCAAGGCCACC P6-3 GACTTCCATCATAGTTACGGCCATGACTCCTCAAGCAAGCAGTGTTAGATCA 134 18 TTCAGCTACCTGAGAGGGACCCTATAACTCTCTACGGCTAACCTGAATGGTC TTCGACATAGTCTAGACCGCCGAGGCCACC P6-4 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 19 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P6-5 GACTTGATGCCGTTATCTTTGGTCAGAATCACCATATCAGCGTTTGCCTCAT 173 20 CTCCCGCAGTCAGTTCATTTTCCAGACCAATTGACCCCTGGGTTTCTACTTT CACTTCCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCTCTGCTGCCATA TTGGTATGGGCAACACC P6-6 GACTTCCATCATAGTTATGGCCATGACTATTCTAGCTAGCAGTGATAAATCA 135 21 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGCCCGCCAAGGCCACC P6-7 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 22 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACAGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P6-8 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 1 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P6-9 Sequencing failed 0 P6-10 GACTTGATGCCGATATCATTAGTCAAAATCACCAAATCAGCGCTCGCCACAT 172 24 CTTCCGCAGTCAGATCATTTTCCAGACCAATTGACCCCTGGGTTTCTACTTT CACTTCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCTTCTGCTGCCATAT AGGTATGGGCAGCACC P6-11 GACTCACCCAGCATCACAGCATCGCGGGCCCCCCATTTATCAGCACGGTAAA 160 25 CAGTTTCTTAAAGCTGTCGCCGCCGTGGTTGGTCATCAATTCCACGCCCGGT AATTCGTCATGCAGACGCTCATCTAATGCCATAGCCGCTGATTGTGTATCAG CACC P6-12 GACTTGATGTCGATATCTTTGGTCAGAATCACCATATCAGCGCTCGCCACAT 173 26 CTTCCGCAATCAGTTCATTTTCCAGACCAATTGATGCCTGGGTTTCTACTTT CACTTCCCAGCCTTTCGCTTTCGCGGCACTTTTCAGCGCCTCTGCTGCCATA TAGGTATGGGCAACACC P7-1 GACTTCCATCATAGTTATGGCCATGACTACTCTACCTAGCAGTGTTAAATCA 135 27 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P7-2 GACTTCAATCATAGTCATGGCTATGACTACTCCAGCTAGCAGTGTTAAATCA 135 28 TTCAGCTACCTGGGAGTGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTTCGCCAAGGCCACC P7-3 GACTTCCATCATAGTTATGACCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 29 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGCCCGCCAAGGCCACC P7-4 GACTTCCATCATAGTTTTGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 30 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTTTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P7-5 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 31 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTTTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P7-6 GACTTCCATCTTAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 32 TTCAGCTACCTGAGAGGGGCCCTTATAACTCTCTACTGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P7-7 GACTTGATGCCGATATCTTTGGCAGAATCACCATATCAGCGCTCGCACATCT 171 33 TCCGCAGTCAGTTCATTATCCAGACTGATTGACCCCTGGGTTTCTACTTTCA CTTCCCAGCCTTTCGCCTTCGCGGCACTTTCCAGCGCCTCTGCTGCCATATA GGTATGGGCAACACC P7-8 GACTTCCATCATAGTTACGGCCATGACTACTCGAGCTAGCAGTGTTAAATCA 135 34 TTCAGCTACCTGAGAGGGGACCTTTTAACTCTCTACGGCTAACCTGTATGGA CTACGACAAAGTCTAGTCAGCCAAGGCCACC P7-9 GACTTGATGCCGATATCTTTGGCAGAATCACCATATCAGCGCTCGCACATCT 171 35 TCCGCAGTCAGTTCATTATCCAGACTGATTGACCCCTGGGTTTCTACTTTCA CTTCCCAGCCTTTCGCCTTCGCGGCACTTTCCAGCGCCTCTGCTGCCATATA GGTATGGGCAACACC P7-10 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAGATCA 135 36 TTCAGCCACCTGAGAGGGGCCCCTAAAACTCTCTACGGCTGACCTGAATGGA CTACGACATAGTTTAGTTCACCAAGGCCACC P7-11 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 37 TTCAGCTACCTGAGAGGGACCCCTATGACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCAGCCAAGGCCACC P7-12 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 120 38 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAAGAGC GCTTCCTCAGGCCACC P8-1 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 39 TTCAGCTACCTGAGATGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P8-2 GACTTCCATCATCGTTATGGCCATGGCTACTCTAGCTAGCAGTGTTAAATCA 135 40 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGTCTAACCGGAATGGA CTACGACATAGTCTAGTCCGCCAAGCCCACC P8-3 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 1 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P8-4 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 1 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P8-5 GACTTGATGCCGATATCTTTGGTCAGAATCACCATATCAGCGCTCGCCACAT 173 43 CTTCCGCAGTCAGTTCATTTTCTAGACCAATTGACCCCTGGGTTTCTACTTT CACTTCCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCTCTGCTGCCATA TAGGTATGGGCAACACC P8-6 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 44 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACTACATAGTCTAGTCCGCCAAGGCCACC P8-7 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 1 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P8-8 GACTTGATGCCGATATCCTTGCTCAGGATCACCGTATCAGCGCTCGCCACAT 173 46 CTTCGGCAGTCAGTTCATTTTCCATACCAACTGGCCCCTGGGTTTCTACTTT CGCGTCCCAGCCTTTCGCTTTCGTGGCACTTTCCAGCATCTCTGCTGCCATA TAGGTATGTGCAACACC P8-9 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGTAGTGTTAAATCA 135 47 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P8-10 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTGAATCA 135 48 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAACGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P8-11 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTAAATCAT 134 49 TCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGCAC TACGACATAGTCTAGTCCGCACAGGCCACC P8-12 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 50 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGGATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P9-1 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 1 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P9-2 Sequencing failed 0 P9-3 GACTTGACGCCGACATCTATGGTCAGAACCACCAAATCAGTGCTCGCCTCAT 173 52 CTTCTGCAGTAAGTTCATTTTCCAGACCAATCGACCCCTGGGTTTCTTCTTT CTCTTCCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCCCTGCTGCCATA TAGGCATGGGTTACACC P9-4 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 53 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTATGGCTAACCTGAATGGA CTACGACTTAGTCTAGTCCGCCATGGCCACC P9-5 Sequencing failed 0 P9-6 GACTTCCAGCATAGTTATCGCCAAGACTACTCTAGCTAGCATTGTTAAATCA 159 54 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATTGTCTAGTCCGCCAAGGCCACCTGAAGAGCGCTTCCTCAGCCC ACC P9-7 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 55 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAGCCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P9-8 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 56 TTCAGCTACCTGAGGGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P9-9 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 1 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P9-10 Sequencing failed 0 P9-11 GACTTGATGCAGGTATCTTTGGTCGGAACCACCATATCAACGCTCTCCACAT 173 58 CCTCCGCAGTCAGCACATTATCCAGACCAATTGACCCCTGTGTTTCTACTAT CTCTTCCCCACCTTTCGCTTTCGCGGCGCTTTCCTGCGCCTCTGCCGCCATA TAGGTATGGGCAACACC P9-12 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGAGTTAATCAT 134 59 TCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCAAACCTGAATGGAC TACGACATAGTCTAGTCCGCCAAGGCCACC P10-1 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 60 TTCAGCTTCCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P10-2 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCA 135 61 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAAAGGT CTACGACATAGTCTAGTCCGCCAAGGCCACC P10-3 Sequencing failed 0 P10-4 GACTTCCATCATAGTTACGGCCATGACCACTATAGCTAGCAGTGTTAAATCA 134 62 TTAAGCTACCTAAGAGGGGCCCCCTTAACTATATACGGCTAACCTGAAGGGC TACGACATAGTCTAGTCCGCCAAGGCCACC P10-5 Sequencing failed 0 P10-6 Sequencing failed 0 P10-7 GACTTCCATCATAGTTATGCCCTTGACTACTCTAGCTAGCAGTGTTAAATCA 135 63 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P10-8 GACTCACCCAGCACCACAGCAACACGGGCTACCCATTCATCAGCCTGGGCAG 161 64 ACTGTTTCTTAAAGTTGCCGACGCCGCGCTTGGTCATCAATCTCACGTCCGG TAATTCATCACTCAGACGCTCTGCTAATGCCATAGCCGCAGATTGTGTATCA GCACC P10-9 GACTTGATGCCGATATCTATGGTCAGGATCACCATTACGTCGCTCGCCACAT 173 65 CTTTCGCAGTCAGTTCATTTTCCAGGCCTATTGACCCCTGGGTTTCTATTAT CACTTCCCATCCTTTCGCATTCGCGGTACTTTCCAGCGCCTCTGCCGCCAAA TAGGTGTAGACTACACC P10-10 Sequencing failed 0 P10-11 GACTTCCATCATAATTAAGGCCATGACTACTCAAGCTAGCAGTGTTAAATCG 135 66 TTCAGTCGCCTAAGAGGGGACACTTTAACTCTCTACGGCTAACCTGTATCGA CTACGACTTAGTCTAGTCTGCCAAGGCCACC P10-12 GACTTGATGCCGATATCTTCGGTCAGAATCACCATATCAGCGCTCACAACAT 172 67 CTTCACCAGTCAGTAGATTTTCCAGACCAATTGACCCGTGGGCTTCTACTTT CACTTCCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCTCTGCTGCCTAT AGGCATGGGCAACACC P11-1 Sequencing failed 0 P11-2 Sequencing failed 0 P11-3 GACTTGATGCCGATATCTCTGGTCAGAATCACCATATCAGCGCTCGCCATAT 173 68 CTTCCGCAGTCAGTTCATTTTCCAAACCAATTGACCCATGGGTTTCTACCTT CACTTCCCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCTCTGCTGCCATA TAGTTATGGGCAACACC P11-4 GACTTCCATCATAGTTACGGCCACGTCTACTCTAGCTTGCAGTGTCAAATCA 62 69 ATCAGCCACC P11-5 Sequencing failed 0 P11-6 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAATGTTAAATCA 135 70 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGCATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P11-7 GACTTGATGACGATATCTTTGGTCGGAATCACCATATCAATGCTCGCCACAT 196 71 CTTCCGCGGTAAGTTCATCTTCCAGACAATTGACCCCTGGGTTACTACTATC ACTTCCCTGCCTTACGCTTTGCGGCACTTACCAGCGCCTCCATTGCCATATA GGAATGGGCAACACCTGAAGAGCGCTTCGTCGAGGCCACC P11-8 Sequencing failed 0 P11-9 Sequencing failed 0 P11-10 GACTTCCATCATAGATATGGCCAAGACTAGTCTAGCTATTAGTGATAAATCA 135 72 TTCAACTACCTGAGAGGGGCCCCCATTACTCACTACGGCTAACCTGAACGGA GTACGGCATTGTCTAGTCTGCCAAGGCCACC P11-11 GACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTGAATCA 135 73 TTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGA CTACGACATAGTCTAGTCCGCCAAGGCCACC P11-12 GACTTGATGCCGATATCTTAGGTCAGAATCACCATATCAGTGATCGCAACAT 173 74 CTTCCGCAGACAATTCATTTTCCAAACCAATTGACCCCTGGGTTTCTACTTT CGCTTCTCAGCCTTTCGCTTTCGCGGCACTTTCCAGCGCCTCTGCTGTCATA AAGGTATGGGCAACACC Consensus GACTTCCATCATACGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATC 80 ATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGG ACTACGACATAGTCTAGTCCGCCAAGGCCACCATATAGGTATGGGCAACACC YRMMGAGCGCTTCGTCGAGGCCACC

Subsequently, a mutant SGP library is created to include SGP mutants that exhibit differential levels of activity. In addition, the library can be expanded to include more SGP mutants once more SGP mutant clones are screened and sequenced.

The strength of selected subgenomic promoters from the library were tested to validate that these promoters indeed have differential levels of activity. Each promoter was cloned into an individual vector driving the expression of the florescent protein mKate. The resulting vectors were amplified by inoculating each individual clones in 10 ml of Terrific Broth media (1:100 dilution from bacteria stock) supplemented with 100 ug/mL of carbenicillin. The cultures were incubated for 16 hours (overnight) at 37° C. on a shaker (225 rpm). Each vector was purified using QIAGEN® Plasmid Plus Midi Kit (Qiagen, Germany) and the concentration of each vector after purification was measured.

A549 cells were transfected with each of the purified vectors for expression of mKate driven by the subgenomic promoters. The expression level of mKate is directly proportional to the strength of the subgenomic promoters. A549 cells were grown to 70% confluency prior to transfection, and the cells were then trypsinized to obtain single cell suspension. One million cells were seeded into each well of a 24 well plates in 500 μL of DMEM/10% FBS.

To transfect the A549 cells with the purified vectors, 750 ng of each purified vector were mixed with 2.25 μL of ViaFect™ Transfection Reagent (Promega, USA) in 30 μL of OptiMeM, and added to the cells prepared in 24 well plate.

The cells were then incubated at 37° C. with 5% CO2 for 48 hours. The cells in each well were trypsinized and washed once with PBS. The cells were resuspended in the FACS buffer (1% BSA, 0.1% NaN3 sodium azide in PBS), and analyzed with LSR Fortessa (BD Biosciences, USA) for mKate expression. Geometric mean of mKate expression were calculated in mVenus positive cells, and relative mKate expression level was graphed. The results indicate that subgenomic mutant promoter strength vary between 161% of WT (P8-3) and 4% of WT. (FIG. 1B)

Example 2: Optimization of Antibody Expression by Expression Cassette Driven by Optimized Subgenomic Promoter Pair from the Library

The SGP mutants identified in the library are used to tune expression level of multiple transgenes from a single alphavirus. The transgenes can be therapeutic molecules, e.g., cytokines, peptides, inhibitors. Moreover, the SGP mutants are used to optimize antibody expression, using an antibody expression cassette illustrated as: Seq1_D1-CMV_nsP(1-4)_SGP1 from subgenomic promoter library_Ipilimumab-HC_3′UTR_SGP2 from subgenomic promoter library_Ipilimumab-LC_3′UTR-rb_glob_PA_Seq2. Alternatively, SGP1 can drive the expression of the light chain of Ipilimumab, while SGP2 can drive the expression of the heavy chain of Ipilimumab. In this construct, the expression of Ipilimumab heavy chain was driven by one of the mutant SGPs selected from the SGP mutant library (SGP1), and the expression of Ipilimumab light chain was driven by another one of the mutant SGPs selected from the SGP mutant library (SGP2). Various SGP1/SGP2 pairs are used to express Ipilimumab. The constructs can then be delivered into suitable cells for expression of the Ipilimumab. The levels of functional Ipilimumab expressed by the expression cassette with different pairs of SGP1 and SGP2 are measured and compared. The expression cassette with SGP1/SGP2 pair that produces the highest level of Ipilimumab is selected for future Ipilimumab production. Likewise, the method is used to select expression cassettes for other antibodies, as well as other molecules, such as therapeutic molecules or detectable molecules.

OTHER EMBODIMENTS

1. An engineered subgenomic promoter library, comprising a plurality of promoters, wherein each promoter comprises a nucleotide sequence of

GACTX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀ X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉ X₄₀X₄₁X₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅X₅₆X₅₇X₅₈ X₅₉X₆₀X₆₁X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇ X₇₈X₇₉X₈₀X₈₁X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄X₉₅X₉₆ X₉₇X₉₈X₉₉X₁₀₀X₁₀₁X₀₁₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉X₁₁₀X₁₁₁ X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉X₁₂₀X₁₂₁X₁₂₂X₁₂₃X₁₂₄X₁₂₅ X₁₂₆X₁₂₇X₁₂₈X₁₂₉X₁₃₀X₁₃₁X₁₃₂X₁₃₃X₁₃₄X₁₃₅X₁₃₆X₁₃₇X₁₃₈X₁₃₉ X₁₄₀X₁₄₁X₁₄₂X₁₄₃X₁₄₄X₁₄₅X₁₄₆X₁₄₇X₁₄₈X₁₄₉X₁₅₀X₁₅₁X₁₅₂X₁₅₃ X₁₅₄X₁₅₅X₁₅₆X₁₅₇X₁₅₈X₁₅₉X₁₆₀X₁₆₁X₁₆₂X₁₆₃X₁₆₄X₁₆₅X₁₆₆X₁₆₇ X₁₆₈X₁₆₉X₁₇₀X₁₇₁X₁₇₂X₁₇₃X₁₇₄X₁₇₅X₁₇₆X₁₇₇X₁₇₈X₁₇₉X₁₈₀X₁₈₁ X₁₈₂X₁₈₃X₁₈₄X₁₈₅X₁₈₆X₁₈₇X₁₈₈X₁₈₉X₁₉₉X₂₀₀X₂₀₁X₂₀₁X₂₀₃X₂₀₄ X₂₀₅X₂₀₆X₂₀₇X₂₀₈X₂₀₉X₂₁₀X₂₁₁X₂₁₂X₂₁₃X₂₁₄X₂₁₅X₂₁₆X₂₁₇X₂₁₈ X₂₁₉X₂₂₀X₂₂₁X₂₂₂X₂₂₃X₂₂₄X₂₂₅X₂₂₆X₂₂₇X₂₂₈X₂₂₉X₂₃₀X₂₃₁X₂₃₂ X₂₃₃X₂₃₄,

wherein X₁ is absent or present, and when X₁ is present, X₁=C; wherein X₂ is absent or present, and when X₂ is present, X₂=A; wherein X₃=T or C; wherein X₄=C or G; wherein X₅=C or A; wherein X₆=C, T or A; wherein X₇=T, G or C; wherein X₈=C, T or A; wherein X₉ is absent or present, and when X₉ is present, X₉=C or A; wherein X₁₀ is absent or present, and when X₁₀ is present, X₁₀=G; wherein X₁₁ is absent or present, and when X₁₁ is present, X₁₁=T, A or G; wherein X₁₂ is absent or present, and when X₁₂ is present, X₁₂=T or C; wherein X₁₃ is absent or present, and when X₁₃ is present, X₁₃=A or C; wherein X₁₄=G, T or A; wherein X₁₅=T, A or C; wherein X₁₆=T, C or A; wherein X₁₇=A, T or C; wherein X₁₈=T, C or A; wherein X₁₉=G, C or A; wherein X₂₀=G, C or A; wherein X₂₁ is absent or present, and when X₂₁ is present, X₂₁=C or T; wherein X₂₂ is absent or present, and when X₂₂ is present, X₂₂=A; wherein X₂₃ is absent or present, and when X₂₃ is present, X₂₃=A or T; wherein X₂₄=C or T; wherein X₂₅=A, T or G; wherein X₂₆=T, A, C, or G; wherein X₂₇=G, T or A; wherein X₂₈=A, G, or T; wherein X₂₉=C, T, or G; wherein X₃₀=T, C or A; wherein X₃₁=A, C or G; wherein X₃₂=C, T, or G; wherein X₃₃=T, C or A; wherein X₃₄=C, A, G or T; wherein X₃₅=T, C, G or A; wherein X₃₆=A, T or G; wherein X₃₇ is absent or present, and when X₃₇ is present, X₃₇=T or A; wherein X₃₈ is absent or present, and when X₃₈ is present, X₃₈=C or T; wherein X₃₉ is absent or present, and when X₃₉ is present, X₃₉=A, G or T; wherein X₄₀ is absent or present, and when X₄₀ is present, X₄=G, A or T; wherein X₄₁ is absent or present, and when X₄₁ is present, X₄₁=C or T; wherein X₄₂=G, C or A; wherein X₄₃=C, A or T; wherein X₄₄=T, A or C; wherein X₄₅=A, T or C; wherein X₄₆=G, T, A or C; wherein X₄₇=C or T; wherein X₄₈ is absent or present, and when X₄₈ is present, X₄₈=C or A; wherein X₄₉ is absent or present, and when X₄₉ is present, X₄₉=A or T; wherein X₅₀ is absent or present, and when X₅₀ is present, X₅₀=C or T; wherein X₅₁ is absent or present, and when X₅₁ is present, X₅₁=A or T; wherein X₅₂ is absent or present, and when X₅₂ is present, X₅₂=T or A; wherein X₅₃ is absent or present, and when X₅₃ is present, X₅₃=C; wherein X₅₄ is absent or present, and when X₅₄ is present, X₅₄=T or C; wherein X₅₅ is absent or present, and when X₅₅ is present, X₅₅=T or C; wherein X₅₆ is absent or present, and when X₅₆ is present, X₅₆=C; wherein X₅₇ is absent or present, and when X₅₇ is present, X₅₇=C, T, G or A; wherein X₅₈ is absent or present, and when X₅₈ is present, X₅₈=T; wherein X₅₉ is absent or present, and when X₅₉ is present, X₅₉=A, G or C; wherein X₆₀ is absent or present, and when X₆₀ is present, X₆₀=G, A, T, C; wherein X₆₁ is absent or present, and when X₆₁ is present, X₆₁=T, A, or G; wherein X₆₂ is absent or present, and when X₆₂ is present, X₆₂=G or A; wherein X₆₃ is absent or present, and when X₆₃ is present, X₆₃=T, A, or G; wherein X₆₄ is absent or present, and when X₆₄ is present, X₆₄=T, G, C, or A; wherein X₆₅ is absent or present, and when X₆₅ is present, X₆₅=A or G; wherein X₆₆ is absent or present, and when X₆₆ is present, X₆₆=A or G; wherein X₆₇ is absent or present, and when X₆₇ is present, X₆₇=A, T, or C; wherein X₆₈ is absent or present, and when X₆₈ is present, X₆₈=T, A, C or G; wherein X₆₉ is absent or present, and when X₆₉ is present, X₆₉=C, T, G or A; wherein X₇₀ is absent or present, and when X₇₀ is present, X₇₀=A, T or G; wherein X₇₁ is absent or present, and when X₇₁ is present, X₇₁=T or A; wherein X₇₂ is absent or present, and when X₇₂ is present, X₇₂=T or C; wherein X₇₃ is absent or present, and when X₇₃ is present, X₇₃=C, A or T; wherein X₇₄ is absent or present, and when X₇₄ is present, X₇₄=A, T or C; wherein X₇₅ is absent or present, and when X₇₅ is present, X₇₅=G, A, C or T; wherein X₇₆ is absent or present, and when X₇₆ is present, X₇₆=C or T; wherein X₇₇ is absent or present, and when X₇₇ is present, X₇₇=T, G, C or A; wherein X₇₈ is absent or present, and when X₇₈ is present, X₇₈=A, T or G; wherein X₇₉ is absent or present, and when X₇₉ is present, X₇₉=C, A or G; wherein X₈₀=C or T; wherein X₈₁ is absent or present, and when X₈₁ is present, X₈₁=T or C; wherein X₈₂ is absent or present, and when X₈₂ is present, X₈₂=G, A or T; wherein X₈₃ is absent or present, and when X₈₃ is present, X₈₃=G; wherein X₈₄ is absent or present, and when X₈₄ is present, X₈₄=G; wherein X₈₅ is absent or present, and when X₈₅ is present, X₈₅=C; wherein X₈₆ is absent or present, and when X₈₆ is present, X₈₆=A, G or T; wherein X₈₇ is absent or present, and when X₈₇ is present, X₈₇=G or A; wherein X₈₈ is absent or present, and when X₈₈ is present, X₈₈=A or G; wherein X₈₉ is absent or present, and when X₈₉ is present, X₈₉=G; wherein X₉₀ is absent or present, and when X₉₀ is present, X₉₀=G, T, A, or C; wherein X₉₁=G, T or C; wherein X₉₂=G, T or A; wherein X₉₃=G, A or T; wherein X₉₄=C, A or T; wherein X₉₅=C, T or G; wherein X₉₆=C, A, or G; wherein X₉₇=C, T, A or G; wherein X₉₈=T, C, G or A; wherein X₉₉=A, T, G or C; wherein X₁₀₀ is absent or present, and when X₁₀₀ is present, X₁₀₀=C; wherein X₁₀₁ is absent or present, and when X₁₀₁ is present, X₁₀₁=C; wherein X₁₀₂ is absent or present, and when X₁₀₂ is present, X₁₀₂=G, T, A or C; wherein X₁₀₃ is absent or present, and when X₁₀₃ is present, X₁₀₃=G, T, or A; wherein X₁₀₄ is absent or present, and when X₁₀₄ is present, X₁₀₄=T, C or G; wherein X₁₀₅ is absent or present, and when X₁₀₅ is present, X₁₀₅=T or G; wherein X₁₀₆ is absent or present, and when X₁₀₆ is present, X₁₀₆=T, A or G; wherein X₁₀₇ is absent or present, and when X₁₀₇ is present, X₁₀₇=T, A or C; wherein X₁₀₈ is absent or present, and when X₁₀₈ is present, X₁₀₈=A, G, T or C; wherein X₁₀₉ is absent or present, and when X₁₀₉ is present, X₁₀₉=A, T or G; wherein X₁₁₀ is absent or present, and when X₁₁₀ is present, X₁₁₀=C, T or G; wherein X₁₁₁ is absent or present, and when X₁₁₁ is present, X₁₁₁=T, or C; wherein X₁₁₂ is absent or present, and when X₁₁₂ is present, X₁₁₂=C, A, T or G; wherein X₁₁₃ is absent or present, and when X₁₁₃ is present, X₁₁₃=T, A or C; wherein X₁₁₄ is absent or present, and when X₁₁₄ is present, X₁₁₄=C, T or A; wherein X₁₁₅ is absent or present, and when X₁₁₅ is present, X₁₁₅=T, A, G or C; wherein X₁₁₆ is absent or present, and when X₁₁₆ is present, X₁₁₆=A or C; wherein X₁₁₇ is absent or present, and when X₁₁₇ is present, X₁₁₇=C, T, G or A; wherein X₁₁₈ is absent or present, and when X₁₁₈ is present, X₁₁₈=G, A, C or T; wherein X₁₁₉ is absent or present, and when X₁₁₉ is present, X₁₁₉=G, C or T; wherein X₁₂₀ is absent or present, and when X₁₂₀ is present, X₁₂₀=C, A or T; wherein X₁₂₁ is absent or present, and when X₁₂₁ is present, X₁₂₁=T, A, C or G; wherein X₁₂₂ is absent or present, and when X₁₂₂ is present, X₁₂₂=A, G, C or T; wherein X₁₂₃ is absent or present, and when X₁₂₃ is present, X₁₂₃=A, G, C or T; wherein X₁₂₄ is absent or present, and when X₁₂₄ is present, X₁₂₄=C or G; wherein X₁₂₅ is absent or present, and when X₁₂₅ is present, X₁₂₅=C or T; wherein X₁₂₆ is absent or present, and when X₁₂₆ is present, X₁₂₆=T, G or C; wherein X₁₂₇ is absent or present, and when X₁₂₇ is present, X₁₂₇=G, C or T; wherein X₁₂₈ is absent or present, and when X₁₂₈ is present, X₁₂₈=G; wherein X₁₂₉ is absent or present, and when X₁₂₉ is present, X₁₂₉=T; wherein X₁₃₀ is absent or present, and when X₁₃₀ is present, X₁₃₀=C; wherein X₁₃₁ is absent or present, and when X₁₃₁ is present, X₁₃₁=A; wherein X₁₃₂ is absent or present, and when X₁₃₂ is present, X₁₃₂=T; wherein X₁₃₃ is absent or present, and when X₁₃₃ is present, X₁₃₃=C; wherein X₁₃₄ is absent or present, and when X₁₃₄ is present, X₁₃₄=A; wherein X₁₃₅ is absent or present, and when X₁₃₅ is present, X₁₃₅=A; wherein X₁₃₆ is absent or present, and when X₁₃₆ is present, X₁₃₆=T; wherein X₁₃₇ is absent or present, and when X₁₃₇ is present, X₁₃₇=C; wherein X₁₃₈ is absent or present, and when X₁₃₈ is present, X₁₃₈=T; wherein X₁₃₉ is absent or present, and when X₁₃₉ is present, X₁₃₉=C; wherein X₁₄₀ is absent or present, and when X₁₄₀ is present, X₁₄₀=A; wherein X₁₄₁ is absent or present, and when X₁₄₁ is present, X₁₄₁=C; wherein X₁₄₂ is absent or present, and when X₁₄₂ is present, X₁₄₂=G; wherein X₁₄₃ is absent or present, and when X₁₄₃ is present, X₁₄₃=T; wherein X₁₄₄ is absent or present, and when X₁₄₄ is present, X₁₄₄=C; wherein X₁₄₅ is absent or present, and when X₁₄₅ is present, X₁₄₅=C; wherein X₁₄₆ is absent or present, and when X₁₄₆ is present, X₁₄₆=A, G, C or T; wherein X₁₄₇ is absent or present, and when X₁₄₇ is present, X₁₄₇=A, T, C or G; wherein X₁₄₈ is absent or present, and when X₁₄₈ is present, X₁₄₈=T, A, C or G; wherein X₁₄₉ is absent or present, and when X₁₄₉ is present, X₁₄₉=G, C, or A; wherein X₁₅₀ is absent or present, and when X₁₅₀ is present, X₁₅₀=G, C, A or T; wherein X₁₅₁ is absent or present, and when X₁₅₁ is present, X₁₅₁=A, C or T; wherein X₁₅₂ is absent or present, and when X₁₅₂ is present, X₁₅₂=C, G or T; wherein X₁₅₃ is absent or present, and when X₁₅₃ is present, X₁₅₃=T or C; wherein X₁₅₄ is absent or present, and when X₁₅₄ is present, X₁₅₄=A, T, G or C; wherein X₁₅₅ is absent or present, and when X₁₅₅ is present, X₁₅₅=C or T; wherein X₁₅₆ is absent or present, and when X₁₅₆ is present, X₁₅₆=G, T, A or C; wherein X₁₅₇ is absent or present, and when X₁₅₇ is present, X₁₅₇=A or G; wherein X₁₅₈ is absent or present, and when X₁₅₈ is present, X₁₅₈=C or T; wherein X₁₅₉ is absent or present, and when X₁₅₉ is present, X₁₅₉=A, T or G; wherein X₁₆₀ is absent or present, and when X₁₆₀ is present, X₁₆₀=T, A, C or G; wherein X₁₆₁ is absent or present, and when X₁₆₁ is present, X₁₆₁=A or T; wherein X₁₆₂ is absent or present, and when X₁₆₂ is present, X₁₆₂=G, T or A; wherein X₁₆₃ is absent or present, and when X₁₆₃ is present, X₁₆₃=A; wherein X₁₆₄ is absent or present, and when X₁₆₄ is present, X₁₆₄=C; wherein X₁₆₅ is absent or present, and when X₁₆₅ is present, X₁₆₅=G or A; wherein X₁₆₆ is absent or present, and when X₁₆₆ is present, X₁₆₆=C; wherein X₁₆₇ is absent or present, and when X₁₆₇ is present, X₁₆₇=T; wherein X₁₆₈ is absent or present, and when X₁₆₈ is present, X₁₆₈=C; wherein X₁₆₉ is absent or present, and when X₁₆₉ is present, X₁₆₉=G or A; wherein X₁₇₀ is absent or present, and when X₁₇₀ is present, X₁₇₀=T, G or A; wherein X₁₇₁ is absent or present, and when X₁₇₁ is present, X₁₇₁=C or T; wherein X₁₇₂ is absent or present, and when X₁₇₂ is present, X₁₇₂=T or C; wherein X₁₇₃ is absent or present, and when X₁₇₃ is present, X₁₇₃=A, G or T; wherein X₁₇₄ is absent or present, and when X₁₇₄ is present, X₁₇₄=G, A or T; wherein X₁₇₅ is absent or present, and when X₁₇₅ is present, X₁₇₅=T, C or A; wherein X₁₇₆ is absent or present, and when X₁₇₆ is present, X₁₇₆=C or T; wherein X₁₇₇ is absent or present, and when X₁₇₇ is present, X₁₇₇=C, A or T; wherein X₁₇₈ is absent or present, and when X₁₇₈ is present, X₁₇₈=G, A or C; wherein X₁₇₉ is absent or present, and when X₁₇₉ is present, X₁₇₉=C, A or T; wherein X₁₈₀ is absent or present, and when X₁₈₀ is present, X₁₈₀=C or T; wherein X₁₈₁ is absent or present, and when X₁₈₁ is present, X₁₈₁=A, C, G or T; wherein X₁₈₂ is absent or present, and when X₁₈₂ is present, X₁₈₂=A, T or C; wherein X₁₈₃ is absent or present, and when X₁₈₃ is present, X₁₈₃=G, T, A, or C; wherein X₁₈₄ is absent or present, and when X₁₈₄ is present, X₁₈₄=G, C or A; wherein X₁₈₅ is absent or present, and when X₁₈₅ is present, X₁₈₅=C or T; wherein X₁₈₆ is absent or present, and when X₁₈₆ is present, X₁₈₆=C, T or A; wherein X₁₈₇ is absent or present, and when X₁₈₇ is present, X₁₈₇=A or G; wherein X₁₈₈ is absent or present, and when X₁₈₈ is present, X₁₈₈=C or T; wherein X₁₈₉ is absent or present, and when X₁₈₉ is present, X₁₈₉=C, A or T; wherein X₁₉₀ is absent or present, and when X₁₉₀ is present, X₁₉₀=A, T, A or G; wherein X₁₉₁ is absent or present, and when X₁₉₁ is present, X₁₉₁=T, G, or A; wherein X₁₉₂ is absent or present, and when X₁₉₂ is present, X₁₉₂=A or T; wherein X₁₉₃ is absent or present, and when X₁₉₃ is present, X₁₉₃=T, or A; wherein X₁₉₄ is absent or present, and when X₁₉₄ is present, X₁₉₄=A or G; wherein X₁₉₅ is absent or present, and when X₁₉₅ is present, X₁₉₅=G, A, or T; wherein X₁₉₆ is absent or present, and when X₁₉₆ is present, X₁₉₆=G or T; wherein X₁₉₇ is absent or present, and when X₁₉₇ is present, X₁₉₇=T, C, or A; wherein X₁₉₈ is absent or present, and when X₁₉₈ is present, X₁₉₈=A or G; wherein X₁₉₉ is absent or present, and when X₁₉₉ is present, X₁₉₉=T or C; wherein X₂₀₀ is absent or present, and when X₂₀₀ is present, X₂₀₀=G, T, A or C; wherein X₂₀₁ is absent or present, and when X₂₀₁ is present, X₂₀₁=G, T or A; wherein X₂₀₂ is absent or present, and when X₂₀₂ is present, X₂₀₂=G, C or A; wherein X₂₀₃ is absent or present, and when X₂₀₃ is present, X₂₀₃=C or T; wherein X₂₀₄ is absent or present, and when X₂₀₄ is present, X₂₀₄=A or T; wherein X₂₀₅ is absent or present, and when X₂₀₅ is present, X₂₀₅=A, C or G; wherein X₂₀₆ is absent or present, and when X₂₀₆ is present, X₂₀₆=C or A; wherein X₂₀₇ is absent or present, and when X₂₀₇ is present, X₂₀₇=A or G; wherein X₂₀₈ is absent or present, and when X₂₀₈ is present, X₂₀₈=C; wherein X₂₀₉ is absent or present, and when X₂₀₉ is present, X₂₀₉=C; wherein X₂₁₀ is absent or present, and when X₂₁₀ is present, X₂₁₀=C or T; wherein X₂₁₁ is absent or present, and when X₂₁₁ is present, X₂₁₁=A or G; wherein X₂₁₂ is absent or present, and when X₂₁₂ is present, X₂₁₂=C or A; wherein X₂₁₃ is absent or present, and when X₂₁₃ is present, X₂₁₃=C or A; wherein X₂₁₄ is absent or present, and when X₂₁₄ is present, X₂₁₄=G; wherein X₂₁₅ is absent or present, and when X₂₁₅ is present, X₂₁₅=A; wherein X₂₁₆ is absent or present, and when X₂₁₆ is present, X₂₁₆=G; wherein X₂₁₇ is absent or present, and when X₂₁₇ is present, X₂₁₇=C; wherein X₂₁₈ is absent or present, and when X₂₁₈ is present, X₂₁₈=G; wherein X₂₁₉ is absent or present, and when X₂₁₉ is present, X₂₁₉=C; wherein X₂₂₀ is absent or present, and when X₂₂₀ is present, X₂₂₀=T; wherein X₂₂₁ is absent or present, and when X₂₂₁ is present, X₂₂₁=T; wherein X₂₂₂ is absent or present, and when X₂₂₂ is present, X₂₂₂=C; wherein X₂₂₃ is absent or present, and when X₂₂₃ is present, X₂₂₃=G; wherein X₂₂₄ is absent or present, and when X₂₂₄ is present, X₂₂₄=T; wherein X₂₂₅ is absent or present, and when X₂₂₅ is present, X₂₂₅=C; wherein X₂₂₆ is absent or present, and when X₂₂₆ is present, X₂₂₆=G; wherein X₂₂₇ is absent or present, and when X₂₂₇ is present, X₂₂₇=A; wherein X₂₂₈ is absent or present, and when X₂₂₈ is present, X₂₂₈=G; wherein X₂₂₉ is absent or present, and when X₂₂₉ is present, X₂₂₉=G; wherein X₂₃₀ is absent or present, and when X₂₃₀ is present, X₂₃₀=C; wherein X₂₃₁ is absent or present, and when X₂₃₁ is present, X₂₃₁=C; wherein X₂₃₂ is absent or present, and when X₂₃₂ is present, X₂₃₂=A; wherein X₂₃₃ is absent or present, and when X₂₃₃ is present, X₂₃₃=C; wherein X₂₃₄ is absent or present, and when X₂₃₄ is present, X₂₃₄=C (SEQ ID NO: 81).

2. The engineered subgenomic promoter library of paragraph 1, wherein the library comprises subgenomic promoter sequences set forth in SEQ ID NO: 1-74.

3. An engineered subgenomic promoter library comprising a plurality of promoters, wherein each promoter comprises a nucleic acid sequence at least 70% identical to nucleic acid sequences of SEQ IDs NO: 1-74 and 81.

4. The engineered subgenomic promoter library of any one of paragraphs 1-3, wherein the promoter is a subgenomic promoter derived from an alphavirus.

5. The engineered subgenomic promoter library of paragraph 4, wherein the alphavirus is Venezuela Equine Encephalitis virus, Semliki Forest virus, or Sindbis virus.

6. The engineered subgenomic promoter library of any one of paragraphs 1-5, wherein each of the subgenomic promoters further comprises restriction endonuclease sites at the 5′ and 3′ ends.

7. The engineered subgenomic promoter library of paragraph 6, wherein the restriction endonuclease site at the 5′ and 3′ ends are SapI sites.

8. The engineered subgenomic promoter library of any one of paragraphs 1-7, wherein the library comprises engineered subgenomic promoters having differential activities.

9. An engineered nucleic acid comprising: (i) a promoter selected from the engineered subgenomic promoter library of any one of paragraphs 1-8; and (ii) a transgene operably linked to the promoter of (i).

10. The engineered nucleic acid of paragraph 9, wherein the first transgene encodes for a therapeutic molecule or a detectable molecule.

11. The engineered nucleic acid of paragraph 10, wherein the first transgene encodes for a heavy chain or a light chain of an antibody.

12. An expression cassette comprising one or more engineered nucleic acid of any one of paragraphs 7-9.

13. The expression cassette of paragraph 12, wherein the expression cassette is an antibody expression cassette.

14. An antibody expression cassette, comprising a first engineered nucleic acid comprising a first engineered subgenomic promoter selected from the engineered subgenomic promoter library of paragraph 1 operably linked to a first transgene, wherein the first transgene encodes a heavy chain of an antibody; and a second engineered nucleic acid comprising a second engineered subgenomic promoter selected from the engineered subgenomic promoter library of paragraph 1 operably linked to a second transgene, wherein the second transgene encodes a light chain of an antibody.

15. A vector comprising one or more engineered nucleic acid of any one of paragraphs 9-11, one or more expression cassette of any one of paragraphs 12-13, or the antibody expression cassette of paragraph 14.

16. The vector of paragraph 15, wherein the vector is a plasmid, RNA replicon, linear double stranded DNA, viral vectors, liposome or nanoparticles.

17. The vector of paragraph 16, wherein the RNA replicon is derived from an alphavirus.

18. The vector of paragraph 17, wherein the one or more engineered nucleic acid is located at the subgenomic region of the RNA replicon.

19. A cell comprising the engineered nucleic acid of any one of paragraphs 9-11, expression cassette of any one of paragraphs 12-13, the antibody expression cassette of paragraph 14, or the vectors of any one of paragraphs 15-18.

20. A method for selecting an antibody expression cassette for optimized production of functional antibody comprising: constructing an antibody expression cassette library comprising a plurality of antibody expression cassette for expression of a heavy chain and a light chain of an antibody, wherein the antibody expression cassette comprises: a first engineered nucleic acid comprising a first engineered subgenomic promoter selected from the engineered subgenomic promoter library of paragraph 1 operably linked to a first transgene, wherein the first transgene encodes a heavy chain of an antibody; and a second engineered nucleic acid comprising a second engineered subgenomic promoter selected from the engineered subgenomic promoter library of paragraph 1 operably linked to a second transgene, wherein the second transgene encodes a light chain of an antibody; delivering the plurality of engineered nucleic acids to a population of cells; culturing the cell under conditions allowing for expression of the heavy chain and the light chain of the antibody; measuring a level of functional antibody comprising the heavy chain and the light chain produced by population of cells; selecting cell(s) expressing optimal level of functional antibody; and determining the nucleic acid sequence of the subgenomic promoter in the first engineered nucleic acid and the nucleic acid sequence of the subgenomic promoter in the second engineered nucleic acid.

21. A method for producing an antibody, comprising: constructing an antibody expression cassette for expression of a heavy chain and a light chain of an antibody, wherein the antibody expression cassette comprises: a first engineered nucleic acid comprising a first engineered subgenomic promoter selected from the engineered subgenomic promoter library of paragraph 1 operably linked to a first transgene, wherein the first transgene encodes a heavy chain of an antibody; and a second engineered nucleic acid comprising a second engineered subgenomic promoter selected from the engineered subgenomic promoter library of paragraph 1 operably linked to a second transgene, wherein the second transgene encodes a light chain of an antibody; delivering the antibody expression cassette to a population of cells or a host animal; culturing the cell or growing the host animal under conditions allowing for expression of the heavy chain and the light chain of the antibody; and harvesting the cultured host cell, culture medium or tissue from the host animal for collection of the antibody.

22. The method of paragraph 21, further comprising purifying the antibody.

23. A method for producing one or more molecules, comprising: constructing an expression cassette for expression of the one or more molecules, wherein the expression cassette comprises one or more engineered nucleic acids of paragraphs 9-11, delivering the expression cassette to a population of cells or a host animal; culturing the cell or growing the host animal under conditions allowing for expression of the one or more molecules; and

harvesting the cultured host cell, culture medium or tissue from the host animal for collection of the molecule.

24. The method of paragraph 23, further comprising purifying the molecule.

25. The method of any one of paragraphs 23 or 24, wherein the molecule encoded by the transgene is a detectable molecule or a therapeutic molecule.

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. 

1. An engineered subgenomic promoter library, comprising a plurality of promoters, wherein each promoter comprises a nucleotide sequence of GACTX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀ X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉ X₄₀X₄₁X₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅X₅₆X₅₇X₅₈ X₅₉X₆₀X₆₁X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇ X₇₈X₇₉X₈₀X₈₁X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄X₉₅X₉₆ X₉₇X₉₈X₉₉X₁₀₀X₁₀₁X₀₁₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉X₁₁₀X₁₁₁ X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉X₁₂₀X₁₂₁X₁₂₂X₁₂₃X₁₂₄X₁₂₅ X₁₂₆X₁₂₇X₁₂₈X₁₂₉X₁₃₀X₁₃₁X₁₃₂X₁₃₃X₁₃₄X₁₃₅X₁₃₆X₁₃₇X₁₃₈X₁₃₉ X₁₄₀X₁₄₁X₁₄₂X₁₄₃X₁₄₄X₁₄₅X₁₄₆X₁₄₇X₁₄₈X₁₄₉X₁₅₀X₁₅₁X₁₅₂X₁₅₃ X₁₅₄X₁₅₅X₁₅₆X₁₅₇X₁₅₈X₁₅₉X₁₆₀X₁₆₁X₁₆₂X₁₆₃X₁₆₄X₁₆₅X₁₆₆X₁₆₇ X₁₆₈X₁₆₉X₁₇₀X₁₇₁X₁₇₂X₁₇₃X₁₇₄X₁₇₅X₁₇₆X₁₇₇X₁₇₈X₁₇₉X₁₈₀X₁₈₁ X₁₈₂X₁₈₃X₁₈₄X₁₈₅X₁₈₆X₁₈₇X₁₈₈X₁₈₉X₁₉₉X₂₀₀X₂₀₁X₂₀₁X₂₀₃X₂₀₄ X₂₀₅X₂₀₆X₂₀₇X₂₀₈X₂₀₉X₂₁₀X₂₁₁X₂₁₂X₂₁₃X₂₁₄X₂₁₅X₂₁₆X₂₁₇X₂₁₈ X₂₁₉X₂₂₀X₂₂₁X₂₂₂X₂₂₃X₂₂₄X₂₂₅X₂₂₆X₂₂₇X₂₂₈X₂₂₉X₂₃₀X₂₃₁X₂₃₂ X₂₃₃X₂₃₄,

wherein X₁ is absent or present, and when X₁ is present, X₁=C; wherein X₂ is absent or present, and when X₂ is present, X₂=A; wherein X₃=T or C; wherein X₄=C or G; wherein X₅=C or A; wherein X₆=C, T or A; wherein X₇=T, G or C; wherein X₈=C, T or A; wherein X₉ is absent or present, and when X₉ is present, X₉=C or A; wherein X₁₀ is absent or present, and when X₁₀ is present, X₁₀=G; wherein X₁₁ is absent or present, and when X₁₁ is present, X₁₁=T, A or G; wherein X₁₂ is absent or present, and when X₁₂ is present, X₁₂=T or C; wherein X₁₃ is absent or present, and when X₁₃ is present, X₁₃=A or C; wherein X₁₄=G, T or A; wherein X₁₅=T, A or C; wherein X₁₆=T, C or A; wherein X₁₇=A, T or C; wherein X₁₈=T, C or A; wherein X₁₉=G, C or A; wherein X₂₀=G, C or A; wherein X₂₁ is absent or present, and when X₂₁ is present, X₂₁=C or T; wherein X₂₂ is absent or present, and when X₂₂ is present, X₂₂=A; wherein X₂₃ is absent or present, and when X₂₃ is present, X₂₃=A or T; wherein X₂₄=C or T; wherein X₂₅=A, T or G; wherein X₂₆=T, A, C, or G; wherein X₂₇=G, T or A; wherein X₂₈=A, G, or T; wherein X₂₉=C, T, or G; wherein X₃₀=T, C or A; wherein X₃₁=A, C or G; wherein X₃₂=C, T, or G; wherein X₃₃=T, C or A; wherein X₃₄=C, A, G or T; wherein X₃₅=T, C, G or A; wherein X₃₆=A, T or G; wherein X₃₇ is absent or present, and when X₃₇ is present, X₃₇=T or A; wherein X₃₈ is absent or present, and when X₃₈ is present, X₃₈=C or T; wherein X₃₉ is absent or present, and when X₃₉ is present, X₃₉=A, G or T; wherein X₄₀ is absent or present, and when X₄₀ is present, X₄₀=G, A or T; wherein X₄₁ is absent or present, and when X₄₁ is present, X₄₁=C or T; wherein X₄₂=G, C or A; wherein X₄₃=C, A or T; wherein X₄₄=T, A or C; wherein X₄₅=A, T or C; wherein X₄₆=G, T, A or C; wherein X₄₇=C or T; wherein X₄₈ is absent or present, and when X₄₈ is present, X₄₈=C or A; wherein X₄₉ is absent or present, and when X₄₉ is present, X₄₉=A or T; wherein X₅₀ is absent or present, and when X₅₀ is present, X₅₀=C or T; wherein X₅₁ is absent or present, and when X₅₁ is present, X₅₁=A or T; wherein X₅₂ is absent or present, and when X₅₂ is present, X₅₂=T or A; wherein X₅₃ is absent or present, and when X₅₃ is present, X₅₃=C; wherein X₅₄ is absent or present, and when X₅₄ is present, X₅₄=T or C; wherein X₅₅ is absent or present, and when X₅₅ is present, X₅₅=T or C; wherein X₅₆ is absent or present, and when X₅₆ is present, X₅₆=C; wherein X₅₇ is absent or present, and when X₅₇ is present, X₅₇=C, T, G or A; wherein X₅₈ is absent or present, and when X₅₈ is present, X₅₈=T; wherein X₅₉ is absent or present, and when X₅₉ is present, X₅₉=A, G or C; wherein X₆₀ is absent or present, and when X₆₀ is present, X₆₀=G, A, T, C; wherein X₆₁ is absent or present, and when X₆₁ is present, X₆₁=T, A, or G; wherein X₆₂ is absent or present, and when X₆₂ is present, X₆₂=G or A; wherein X₆₃ is absent or present, and when X₆₃ is present, X₆₃=T, A, or G; wherein X₆₄ is absent or present, and when X₆₄ is present, X₆₄=T, G, C, or A; wherein X₆₅ is absent or present, and when X₆₅ is present, X₆₅=A or G; wherein X₆₆ is absent or present, and when X₆₆ is present, X₆₆=A or G; wherein X₆₇ is absent or present, and when X₆₇ is present, X₆₇=A, T, or C; wherein X₆₈ is absent or present, and when X₆₈ is present, X₆₈=T, A, C or G; wherein X₆₉ is absent or present, and when X₆₉ is present, X₆₉=C, T, G or A; wherein X₇₀ is absent or present, and when X₇₀ is present, X₇₀=A, T or G; wherein X₇₁ is absent or present, and when X₇₁ is present, X₇₁=T or A; wherein X₇₂ is absent or present, and when X₇₂ is present, X₇₂=T or C; wherein X₇₃ is absent or present, and when X₇₃ is present, X₇₃=C, A or T; wherein X₇₄ is absent or present, and when X₇₄ is present, X₇₄=A, T or C; wherein X₇₅ is absent or present, and when X₇₅ is present, X₇₅=G, A, C or T; wherein X₇₆ is absent or present, and when X₇₆ is present, X₇₆=C or T; wherein X₇₇ is absent or present, and when X₇₇ is present, X₇₇=T, G, C or A; wherein X₇₈ is absent or present, and when X₇₈ is present, X₇₈=A, T or G; wherein X₇₉ is absent or present, and when X₇₉ is present, X₇₉=C, A or G; wherein X₈₀=C or T; wherein X₈₁ is absent or present, and when X₈₁ is present, X₈₁=T or C; wherein X₈₂ is absent or present, and when X₈₂ is present, X₈₂=G, A or T; wherein X₈₃ is absent or present, and when X₈₃ is present, X₈₃=G; wherein X₈₄ is absent or present, and when X₈₄ is present, X₈₄=G; wherein X₈₅ is absent or present, and when X₈₅ is present, X₈₅=C; wherein X₈₆ is absent or present, and when X₈₆ is present, X₈₆=A, G or T; wherein X₈₇ is absent or present, and when X₈₇ is present, X₈₇=G or A; wherein X₈₈ is absent or present, and when X₈₈ is present, X₈₈=A or G; wherein X₈₉ is absent or present, and when X₈₉ is present, X₈₉=G; wherein X₉₀ is absent or present, and when X₉₀ is present, X₉₀=G, T, A, or C; wherein X₉₁=G, T or C; wherein X₉₂=G, T or A; wherein X₉₃=G, A or T; wherein X₉₄=C, A or T; wherein X₉₅=C, T or G; wherein X₉₆=C, A, or G; wherein X₉₇=C, T, A or G; wherein X₉₈=T, C, G or A; wherein X₉₉=A, T, G or C; wherein X₁₀₀ is absent or present, and when X₁₀₀ is present, X₁₀₀=C; wherein X₁₀₁ is absent or present, and when X₁₀₁ is present, X₁₀₁=C; wherein X₁₀₂ is absent or present, and when X₁₀₂ is present, X₁₀₂=G, T, A or C; wherein X₁₀₃ is absent or present, and when X₁₀₃ is present, X₁₀₃=G, T, or A; wherein X₁₀₄ is absent or present, and when X₁₀₄ is present, X₁₀₄=T, C or G; wherein X₁₀₅ is absent or present, and when X₁₀₅ is present, X₁₀₅=T or G; wherein X₁₀₆ is absent or present, and when X₁₀₆ is present, X₁₀₆=T, A or G; wherein X₁₀₇ is absent or present, and when X₁₀₇ is present, X₁₀₇=T, A or C; wherein X₁₀₈ is absent or present, and when X₁₀₈ is present, X₁₀₈=A, G, T or C; wherein X₁₀₉ is absent or present, and when X₁₀₉ is present, X₁₀₉=A, T or G; wherein X₁₁₀ is absent or present, and when X₁₁₀ is present, X₁₁₀=C, T or G; wherein X₁₁₁ is absent or present, and when X₁₁₁ is present, X₁₁₁=T, or C; wherein X₁₁₂ is absent or present, and when X₁₁₂ is present, X₁₁₂=C, A, T or G; wherein X₁₁₃ is absent or present, and when X₁₁₃ is present, X₁₁₃=T, A or C; wherein X₁₁₄ is absent or present, and when X₁₁₄ is present, X₁₁₄=C, T or A; wherein X₁₁₅ is absent or present, and when X₁₁₅ is present, X₁₁₅=T, A, G or C; wherein X₁₁₆ is absent or present, and when X₁₁₆ is present, X₁₁₆=A or C; wherein X₁₁₇ is absent or present, and when X₁₁₇ is present, X₁₁₇=C, T, G or A; wherein X₁₁₈ is absent or present, and when X₁₁₈ is present, X₁₁₈=G, A, C or T; wherein X₁₁₉ is absent or present, and when X₁₁₉ is present, X₁₁₉=G, C or T; wherein X₁₂₀ is absent or present, and when X₁₂₀ is present, X₁₂₀=C, A or T; wherein X₁₂₁ is absent or present, and when X₁₂₁ is present, X₁₂₁=T, A, C or G; wherein X₁₂₂ is absent or present, and when X₁₂₂ is present, X₁₂₂=A, G, C or T; wherein X₁₂₃ is absent or present, and when X₁₂₃ is present, X₁₂₃=A, G, C or T; wherein X₁₂₄ is absent or present, and when X₁₂₄ is present, X₁₂₄=C or G; wherein X₁₂₅ is absent or present, and when X₁₂₅ is present, X₁₂₅=C or T; wherein X₁₂₆ is absent or present, and when X₁₂₆ is present, X₁₂₆=T, G or C; wherein X₁₂₇ is absent or present, and when X₁₂₇ is present, X₁₂₇=G, C or T; wherein X₁₂₈ is absent or present, and when X₁₂₈ is present, X₁₂₈=G; wherein X₁₂₉ is absent or present, and when X₁₂₉ is present, X₁₂₉=T; wherein X₁₃₀ is absent or present, and when X₁₃₀ is present, X₁₃₀=C; wherein X₁₃₁ is absent or present, and when X₁₃₁ is present, X₁₃₁=A; wherein X₁₃₂ is absent or present, and when X₁₃₂ is present, X₁₃₂=T; wherein X₁₃₃ is absent or present, and when X₁₃₃ is present, X₁₃₃=C; wherein X₁₃₄ is absent or present, and when X₁₃₄ is present, X₁₃₄=A; wherein X₁₃₅ is absent or present, and when X₁₃₅ is present, X₁₃₅=A; wherein X₁₃₆ is absent or present, and when X₁₃₆ is present, X₁₃₆=T; wherein X₁₃₇ is absent or present, and when X₁₃₇ is present, X₁₃₇=C; wherein X₁₃₈ is absent or present, and when X₁₃₈ is present, X₁₃₈=T; wherein X₁₃₉ is absent or present, and when X₁₃₉ is present, X₁₃₉=C; wherein X₁₄₀ is absent or present, and when X₁₄₀ is present, X₁₄₀=A; wherein X₁₄₁ is absent or present, and when X₁₄₁ is present, X₁₄₁=C; wherein X₁₄₂ is absent or present, and when X₁₄₂ is present, X₁₄₂=G; wherein X₁₄₃ is absent or present, and when X₁₄₃ is present, X₁₄₃=T; wherein X₁₄₄ is absent or present, and when X₁₄₄ is present, X₁₄₄=C; wherein X₁₄₅ is absent or present, and when X₁₄₅ is present, X₁₄₅=C; wherein X₁₄₆ is absent or present, and when X₁₄₆ is present, X₁₄₆=A, G, C or T; wherein X₁₄₇ is absent or present, and when X₁₄₇ is present, X₁₄₇=A, T, C or G; wherein X₁₄₈ is absent or present, and when X₁₄₈ is present, X₁₄₈=T, A, C or G; wherein X₁₄₉ is absent or present, and when X₁₄₉ is present, X₁₄₉=G, C, or A; wherein X₁₅₀ is absent or present, and when X₁₅₀ is present, X₁₅₀=G, C, A or T; wherein X₁₅₁ is absent or present, and when X₁₅₁ is present, X₁₅₁=A, C or T; wherein X₁₅₂ is absent or present, and when X₁₅₂ is present, X₁₅₂=C, G or T; wherein X₁₅₃ is absent or present, and when X₁₅₃ is present, X₁₅₃=T or C; wherein X₁₅₄ is absent or present, and when X₁₅₄ is present, X₁₅₄=A, T, G or C; wherein X₁₅₅ is absent or present, and when X₁₅₅ is present, X₁₅₅=C or T; wherein X₁₅₆ is absent or present, and when X₁₅₆ is present, X₁₅₆=G, T, A or C; wherein X₁₅₇ is absent or present, and when X₁₅₇ is present, X₁₅₇=A or G; wherein X₁₅₈ is absent or present, and when X₁₅₈ is present, X₁₅₈=C or T; wherein X₁₅₉ is absent or present, and when X₁₅₉ is present, X₁₅₉=A, T or G; wherein X₁₆₀ is absent or present, and when X₁₆₀ is present, X₁₆₀=T, A, C or G; wherein X₁₆₁ is absent or present, and when X₁₆₁ is present, X₁₆₁=A or T; wherein X₁₆₂ is absent or present, and when X₁₆₂ is present, X₁₆₂=G, T or A; wherein X₁₆₃ is absent or present, and when X₁₆₃ is present, X₁₆₃=A; wherein X₁₆₄ is absent or present, and when X₁₆₄ is present, X₁₆₄=C; wherein X₁₆₅ is absent or present, and when X₁₆₅ is present, X₁₆₅=G or A; wherein X₁₆₆ is absent or present, and when X₁₆₆ is present, X₁₆₆=C; wherein X₁₆₇ is absent or present, and when X₁₆₇ is present, X₁₆₇=T; wherein X₁₆₈ is absent or present, and when X₁₆₈ is present, X₁₆₈=C; wherein X₁₆₉ is absent or present, and when X₁₆₉ is present, X₁₆₉=G or A; wherein X₁₇₀ is absent or present, and when X₁₇₀ is present, X₁₇₀=T, G or A; wherein X₁₇₁ is absent or present, and when X₁₇₁ is present, X₁₇₁=C or T; wherein X₁₇₂ is absent or present, and when X₁₇₂ is present, X₁₇₂=T or C; wherein X₁₇₃ is absent or present, and when X₁₇₃ is present, X₁₇₃=A, G or T; wherein X₁₇₄ is absent or present, and when X₁₇₄ is present, X₁₇₄=G, A or T; wherein X₁₇₅ is absent or present, and when X₁₇₅ is present, X₁₇₅=T, C or A; wherein X₁₇₆ is absent or present, and when X₁₇₆ is present, X₁₇₆=C or T; wherein X₁₇₇ is absent or present, and when X₁₇₇ is present, X₁₇₇=C, A or T; wherein X₁₇₈ is absent or present, and when X₁₇₈ is present, X₁₇₈=G, A or C; wherein X₁₇₉ is absent or present, and when X₁₇₉ is present, X₁₇₉=C, A or T; wherein X₁₈₀ is absent or present, and when X₁₈₀ is present, X₁₈₀=C or T; wherein X₁₈₁ is absent or present, and when X₁₈₁ is present, X₁₈₁=A, C, G or T; wherein X₁₈₂ is absent or present, and when X₁₈₂ is present, X₁₈₂=A, T or C; wherein X₁₈₃ is absent or present, and when X₁₈₃ is present, X₁₈₃=G, T, A, or C; wherein X₁₈₄ is absent or present, and when X₁₈₄ is present, X₁₈₄=G, C or A; wherein X₁₈₅ is absent or present, and when X₁₈₅ is present, X₁₈₅=C or T; wherein X₁₈₆ is absent or present, and when X₁₈₆ is present, X₁₈₆=C, T or A; wherein X₁₈₇ is absent or present, and when X₁₈₇ is present, X₁₈₇=A or G; wherein X₁₈₈ is absent or present, and when X₁₈₈ is present, X₁₈₈=C or T; wherein X₁₈₉ is absent or present, and when X₁₈₉ is present, X₁₈₉=C, A or T; wherein X₁₉₀ is absent or present, and when X₁₉₀ is present, X₁₉₀=A, T, A or G; wherein X₁₉₁ is absent or present, and when X₁₉₁ is present, X₁₉₁=T, G, or A; wherein X₁₉₂ is absent or present, and when X₁₉₂ is present, X₁₉₂=A or T; wherein X₁₉₃ is absent or present, and when X₁₉₃ is present, X₁₉₃=T, or A; wherein X₁₉₄ is absent or present, and when X₁₉₄ is present, X₁₉₄=A or G; wherein X₁₉₅ is absent or present, and when X₁₉₅ is present, X₁₉₅=G, A, or T; wherein X₁₉₆ is absent or present, and when X₁₉₆ is present, X₁₉₆=G or T; wherein X₁₉₇ is absent or present, and when X₁₉₇ is present, X₁₉₇=T, C, or A; wherein X₁₉₈ is absent or present, and when X₁₉₈ is present, X₁₉₈=A or G; wherein X₁₉₉ is absent or present, and when X₁₉₉ is present, X₁₉₉=T or C; wherein X₂₀₀ is absent or present, and when X₂₀₀ is present, X₂₀₀=G, T, A or C; wherein X₂₀₁ is absent or present, and when X₂₀₁ is present, X₂₀₁=G, T or A; wherein X₂₀₂ is absent or present, and when X₂₀₂ is present, X₂₀₂=G, C or A; wherein X₂₀₃ is absent or present, and when X₂₀₃ is present, X₂₀₃=C or T; wherein X₂₀₄ is absent or present, and when X₂₀₄ is present, X₂₀₄=A or T; wherein X₂₀₅ is absent or present, and when X₂₀₅ is present, X₂₀₅=A, C or G; wherein X₂₀₆ is absent or present, and when X₂₀₆ is present, X₂₀₆=C or A; wherein X₂₀₇ is absent or present, and when X₂₀₇ is present, X₂₀₇=A or G; wherein X₂₀₈ is absent or present, and when X₂₀₈ is present, X₂₀₈=C; wherein X₂₀₉ is absent or present, and when X₂₀₉ is present, X₂₀₉=C; wherein X₂₁₀ is absent or present, and when X₂₁₀ is present, X₂₁₀=C or T; wherein X₂₁₁ is absent or present, and when X₂₁₁ is present, X₂₁₁=A or G; wherein X₂₁₂ is absent or present, and when X₂₁₂ is present, X₂₁₂=C or A; wherein X₂₁₃ is absent or present, and when X₂₁₃ is present, X₂₁₃=C or A; wherein X₂₁₄ is absent or present, and when X₂₁₄ is present, X₂₁₄=G; wherein X₂₁₅ is absent or present, and when X₂₁₅ is present, X₂₁₅=A; wherein X₂₁₆ is absent or present, and when X₂₁₆ is present, X₂₁₆=G; wherein X₂₁₇ is absent or present, and when X₂₁₇ is present, X₂₁₇=C; wherein X₂₁₈ is absent or present, and when X₂₁₈ is present, X₂₁₈=G; wherein X₂₁₉ is absent or present, and when X₂₁₉ is present, X₂₁₉=C; wherein X₂₂₀ is absent or present, and when X₂₂₀ is present, X₂₂₀=T; wherein X₂₂₁ is absent or present, and when X₂₂₁ is present, X₂₂₁=T; wherein X₂₂₂ is absent or present, and when X₂₂₂ is present, X₂₂₂=C; wherein X₂₂₃ is absent or present, and when X₂₂₃ is present, X₂₂₃=G; wherein X₂₂₄ is absent or present, and when X₂₂₄ is present, X₂₂₄=T; wherein X₂₂₅ is absent or present, and when X₂₂₅ is present, X₂₂₅=C; wherein X₂₂₆ is absent or present, and when X₂₂₆ is present, X₂₂₆=G; wherein X₂₂₇ is absent or present, and when X₂₂₇ is present, X₂₂₇=A; wherein X₂₂₈ is absent or present, and when X₂₂₈ is present, X₂₂₈=G; wherein X₂₂₉ is absent or present, and when X₂₂₉ is present, X₂₂₉=G; wherein X₂₃₀ is absent or present, and when X₂₃₀ is present, X₂₃₀=C; wherein X₂₃₁ is absent or present, and when X₂₃₁ is present, X₂₃₁=C; wherein X₂₃₂ is absent or present, and when X₂₃₂ is present, X₂₃₂=A; wherein X₂₃₃ is absent or present, and when X₂₃₃ is present, X₂₃₃=C; wherein X₂₃₄ is absent or present, and when X₂₃₄ is present, X₂₃₄=C.
 2. The engineered subgenomic promoter library of claim 1, wherein the library comprises subgenomic promoter sequences set forth in SEQ ID NO: 1-74.
 3. An engineered subgenomic promoter library comprising a plurality of promoters, wherein each promoter comprises a nucleic acid sequence at least 70% identical to nucleic acid sequences of SEQ IDs NO: 1-74 and
 81. 4. The engineered subgenomic promoter library of claim 1, wherein the promoter is a subgenomic promoter derived from an alphavirus.
 5. The engineered subgenomic promoter library of claim 4, wherein the alphavirus is Venezuela Equine Encephalitis virus, Semliki Forest virus, or Sindbis virus.
 6. The engineered subgenomic promoter library of claim 1, wherein each of the subgenomic promoters further comprises restriction endonuclease sites at the 5′ and 3′ ends.
 7. (canceled)
 8. The engineered subgenomic promoter library of claim 1, wherein the library comprises engineered subgenomic promoters having differential activities.
 9. An engineered nucleic acid comprising: (i) a promoter selected from the engineered subgenomic promoter library of claim 1; and (ii) a transgene operably linked to the promoter of (i).
 10. The engineered nucleic acid of claim 9, wherein the first transgene encodes for a therapeutic molecule or a detectable molecule.
 11. The engineered nucleic acid of claim 10, wherein the first transgene encodes for a heavy chain or a light chain of an antibody.
 12. An expression cassette comprising one or more engineered nucleic acid of claim
 9. 13. The expression cassette of claim 12, wherein the expression cassette is an antibody expression cassette.
 14. An antibody expression cassette, comprising a first engineered nucleic acid comprising a first engineered subgenomic promoter selected from the engineered subgenomic promoter library of claim 1 operably linked to a first transgene, wherein the first transgene encodes a heavy chain of an antibody; and a second engineered nucleic acid comprising a second engineered subgenomic promoter selected from the engineered subgenomic promoter library of claim 1 operably linked to a second transgene, wherein the second transgene encodes a light chain of an antibody.
 15. A vector comprising one or more engineered nucleic acid of claim
 9. 16. The vector of claim 15, wherein the vector is a plasmid, RNA replicon, linear double stranded DNA, viral vector, liposome, or nanoparticle.
 17. (canceled)
 18. The vector of claim 16, wherein the one or more engineered nucleic acid is located at the subgenomic region of the RNA replicon.
 19. A cell comprising the engineered nucleic acid of claim
 9. 20. A method for selecting an antibody expression cassette for optimized production of functional antibody comprising: constructing an antibody expression cassette library comprising a plurality of antibody expression cassette for expression of a heavy chain and a light chain of an antibody, wherein the antibody expression cassette comprises: a first engineered nucleic acid comprising a first engineered subgenomic promoter selected from the engineered subgenomic promoter library of claim 1 operably linked to a first transgene, wherein the first transgene encodes a heavy chain of an antibody; and a second engineered nucleic acid comprising a second engineered subgenomic promoter selected from the engineered subgenomic promoter library of claim 1 operably linked to a second transgene, wherein the second transgene encodes a light chain of an antibody; delivering the plurality of engineered nucleic acids to a population of cells; culturing the cell under conditions allowing for expression of the heavy chain and the light chain of the antibody; measuring a level of functional antibody comprising the heavy chain and the light chain produced by population of cells; selecting cell(s) expressing optimal level of functional antibody; and determining the nucleic acid sequence of the subgenomic promoter in the first engineered nucleic acid and the nucleic acid sequence of the subgenomic promoter in the second engineered nucleic acid.
 21. A method for producing an antibody, comprising: constructing an antibody expression cassette for expression of a heavy chain and a light chain of an antibody, wherein the antibody expression cassette comprises: a first engineered nucleic acid comprising a first engineered subgenomic promoter selected from the engineered subgenomic promoter library of claim 1 operably linked to a first transgene, wherein the first transgene encodes a heavy chain of an antibody; and a second engineered nucleic acid comprising a second engineered subgenomic promoter selected from the engineered subgenomic promoter library of claim 1 operably linked to a second transgene, wherein the second transgene encodes a light chain of an antibody; delivering the antibody expression cassette to a population of cells or a host animal; culturing the cell or growing the host animal under conditions allowing for expression of the heavy chain and the light chain of the antibody; and harvesting the cultured host cell, culture medium or tissue from the host animal for collection of the antibody.
 22. (canceled)
 23. A method for producing one or more molecules, comprising: constructing an expression cassette for expression of the one or more molecules, wherein the expression cassette comprises one or more engineered nucleic acids of claim 9, delivering the expression cassette to a population of cells or a host animal; culturing the cell or growing the host animal under conditions allowing for expression of the one or more molecules; and harvesting the cultured host cell, culture medium or tissue from the host animal for collection of the molecule. 24.-25. (canceled) 